LncRNAs are defined as RNA transcripts greater than 200 nucleotides in length that have no or limited protein-coding potential. Basal expression of lncRNAs appeared important for various homeostatic processes, like gene imprinting cell differentiation and organogenesis. Moreover, it has been demonstrated that lncRNAs play an important role in tumorigenesis and metastasis. Some lncRNAs were stably detected in exosomes, which are widely found in body fluids. Several studies validated the use of exosomal lncRNAs as minimally invasive diagnostic and prognostic markers in several types of cancers. In addition, exosomal lncRNAs have been associated with drug resistance of tumor cells, suggesting a clinical application in cancer-targeted therapy. Despite the recent increase of studies on exosomal lncRNAs, their clinical significance in cancer diagnosis, prognosis and treatment needs to be fully explored. The methodologies for their detection with high purity and accuracy must be also improved in order to implement their use in clinical routine. This review aims to summarize the main recent technologies available for the isolation of exosomal lncRNAs, their status as a liquid biopsy as well as their future perspectives. The location of a lncRNA is often considered relatively to its neighboring protein-coding gene(s). LncRNA genes overlapping with protein-coding genes are antisense lncRNAs; intronic lncRNAs are located in the introns of protein-coding genes; lncRNA genes in the intergenic genomic loci are termed lincRNA. In addition, some genes encode both protein-coding and lncRNA genes. In recent years, lncRNA were identified to regulate gene expression through various mechanisms. Basal expression of lncRNAs in many tissues regulates various homeostatic processes, including gene imprinting, cell differentiation and organogenesis,5. There is also a strong association between deregulated lncRNA expression and disease evolution. Indeed lncRNAs play an important role in tumorigenesis and metastasis,7. For instance, in liver, gastric and breast cancers, GAS5 and HULC lncRNAs are involved in the control of cell invasion by regulating miRNAs and the interactions between tumor microenvironment, and cancer cells-10. LncRNAs are then expressed abundantly by cancer cells and show greater tissue-specificity compared to protein-coding mRNAs. Thus, expression levels of lncRNAs may represent a powerful biomarker.LncRNAs are classically defined as RNA transcripts greater than 200 nucleotides in length that have no or limited protein-coding potential. To exert their function, lncRNAs interact in both sequence-specific and conformational engagements acting as scaffolds, decoys (molecular sink/miRNA sponge) and enhancer RNAs. Interestingly, lncRNAs can also be packaged into exosomes and act as messengers in cell-to-cell communications. Notably, some lncRNAs are enriched in exosomes, while others are barely present, indicating that lncRNAs are selectively sorted into exosomes. Specific proteins might act as fundamental lncRNAs carriers to control lncRNA release into exosomes. Unfortunately, the exact mechanisms associated with this regulation are not yet fully elucidated. Recently, it has been demonstrated that exosome-derived lncRNAs regulate tumor cell apoptosis, proliferation and migration and induce angiogenesis,15.LncRNAs are localized in either the nucleus or cytoplasm, and can interact with DNA, RNA or proteins. In addition, exosomes modulate the escape of cancer cells from immune cells by releasing immunoregulatory molecules. For example, Qu et al. found a high expression of lncARSR in sunitinib-resistant RCC cells, which were able to disseminate survival skills to other recipient cells via exosomes containing lncARSR. Some studies showed that lncRNAs secreted by tumor-derived exosomes could stimulate the proangiogenic potential of circulating angiogenic cells by increasing expression of both membrane molecules and soluble factors. In this regard, Ma et al. demonstrated a high expression of HOTAIR lncRNA in glioma cells. Interestingly, HOTAIR is packed into exosomes secreted by glioma cells and transmitted to endothelial cells where it stimulates angiogenesis by increasing the expression of the proangiogenic factor VEGFA. Interactions between metastatic cells and their microenvironment via lncRNA-containing exosomes were also demonstrated. High expression of exosomal HOTAIR was correlated with tumor migration and metastasis in several studies-22. In addition, lncRNAs secreted by exosomes can regulate tumor stem cell characteristics. For instance, Xu et al. showed that the GAS5 lncRNA controls human embryonic stem cell self-renewal.The influence of exosomal lncRNAs in the tumor microenvironment has been recently investigated. Cancer and stromal cells use exosomes to modify surrounding cells within tumor microenvironment by transferring ncRNAs and proteins, which activate signaling pathways via receptor-ligand interactions and contribute to tumor progression and metastasis,25. The present review aims to summarize the main recent technologies available for the isolation and detection of exosomal lncRNAs and their status as liquid biopsies. Moreover, it will focus on future perspectives.The relationship between exosomal lncRNA and cancer disease progression as well as exosome isolation from liquid biopsies make exosome-derived lncRNAs excellent candidates as potential diagnostic and prognostic biomarkers of various cancers. Finally, RNA analysis allows the detection of several molecules such as non-coding RNAs, fusion transcripts, splice variants and RNA editing events.In the field of biomarkers, RNA exhibits some advantages over DNA. First, the expression pattern of several RNA molecules, in particular lncRNAs, is highly tissue- or disease-state-specific. In addition, RNA expression is dynamic and fluctuates according to the internal needs of cells. The ideal cancer biomarker must be able to identify the cancer subtype, the stage of the disease, the most efficient therapy and to predict/detect tumor recurrences. Moreover, it should be detected by a noninvasive method applicable into clinical routine. Thanks to their specificity, lncRNAs detectable in body fluids have the potential to meet these expectations, but some essential obstacles still need to be addressed.Liquid biopsy compared to traditional solid biopsy has been widely used as a noninvasive diagnosis and molecular phenotyping technology to detect early cancer states. Liquid biopsy has been also proposed to determine tumor dynamics and can provide diagnostic and prognostic information prior to treatment, during treatment and during progression. These extracellular vesicles are widely found in body fluids, including blood, tears, urine, saliva, milk, ascites, etc.. Cancer cells secrete more exosomes than normal cells, and the biological information of cancer can be directly obtained by analyzing cancer-derived exosomes. Exosomes are involved in critical processes of cancer development, including tumor growth, metastasis, drug resistance and tumor microenvironment,32. Exosomal lncRNAs are differently expressed in many cancers suggesting that they could reflect the physiological and pathological status of their parental cells,33. Moreover several studies reported great differences in the expression profiles of exosomal lncRNAs between healthy people and cancer patients,35, as well as among cancer patients at different stages of the disease. This tumor-association specificity has attracted attention in precision oncology as promising biomarkers for cancer diagnosis as well as ideal prognostic biomarkers for cancer management. Interestingly, Dong et al., following by an incubation and low- speed centrifugation steps. Antibodies against these markers are immobilized on coated beads (as Protein G coated Dynabeads™) which bind the exosomal specific markers , afterwards the beads-exosome complexes are pelleted using magnets. The main advantage over other methods is its selectivity leading to the high purity of exosomes collected. However, this method is expensive and can only be used for small sample volumes and for high concentrated samples. The exosome yield from immunoafinity remains relatively small then reducing the potential for further analysis to low consumption methods. Nonspecific binding and potential detachment of exosomes from beads limit its interest,61.Immunoafinity is based on the expression of specific protein markers expressed at the exosome membrane such as tetraspanins, integrins or adhesion molecules,63. For instance, ELISA-based ExoTEST™, ExoQuick and immunoaffinitive superparamagnetic nanoparticles (ISPN) were proposed for isolating exosomes from various biological fluids-65. ExoQuick kits do not require specific equipment, are regularly updated and the extraction efficiency is gradually improved. Interestingly, a recent study by Niu et al. compared the application of ultracentrifugation, ultrafiltration and polymer-based precipitation for exosomal isolation from human endometrial cells and showed that polymer-based method led to the lowest protein contamination. Another study compared six kits for serum exosome purification  and demonstrated that all of them can isolate exosomes but with variable efficiency and contamination by lipoproteins and albumin. Three of these methods obtained similar yield: Exo-spin, exoEasy and ExoQuick. However, exoEasy or ExoQuick Plus resulted in the highest purity.In the recent years, various commercial kits have been developed to separate and purify exosomes. Continual advances in nanotechnology made possible the development of several Mf-IAC devices that allow the isolation of exosomes with sensitivity and specificity while suppressing non-specific capture-72. Mf-IAC can isolate exosomes from culture medium or body fluids. However, it is mandatory to know previously the surface antigen to target. Mf-F is based on the specific size of exosomes. The pioneer Mf-F devices were pressure- and electrophoresis-driven, which separate exosomes from undesirable particles via a nanoporous membrane with an adjustable pore size. Further on, double-filtration approaches were developed, offering significantly higher throughput and higher purity. The NT system is a multiscale filtration system using nanowires. NT selectively traps specific sizes of particles, and the concept is similar to size exclusion chromatography. Nano-DLD is a pillar-array-based microfluidic device that sorts exosomes in continuous flow by taking advantage of the streamlines that particles are forced to follow due to the physical constrains imposed by the chip design or by the mechanical properties of the device. The viscoelastic flow sorting is a continuous, size based and label-free method where exosome isolation is determined by elastic lift forces acting on particles of different sizes in a viscoelastic medium. This method showed a very high level of purity (> 90%) and good yields. An acoustic isolation system relies on the application of differential of acoustic pressure fields that can separate small particles based on their mechanical properties including compressibility, diameter and density. Interestingly Wu et al. recently established an acoustofluidics technique based on two modules: a cell-removal module and an EV-isolation module, that greatly simplifies the pre-processing of complex samples such as blood. Finally, DEP devices enable exosomes isolation in the presence of a non-uniform electric field. Separation is based on dielectric constant differences that determine how quickly particles move. Advantages of this approach are the few instrumentation requirements and exosome structure remains unaffected.Besides the traditional methodologies described above, microfluidic-based exosome isolation on-a-chip systems have recently emerged. Microfluidic-based exosome isolation approaches can be divided in: (1) systems based on specific surface markers such as microfluidics-based immunoafinity capture (Mf-IAC); (2) techniques related to specific size and density such as microfluidics-based membrane filtration (Mf-F), nanowire-based traps (NTs), nano-sized deterministic lateral displacement (nano-DLD), viscoelastic flow and acoustic isolation; and (3) devices based on other physical properties, such as dielectrophoresis (DEP). Briefly, Mf-IAC consists in targeting exosomal specific surface markers either with selected antibodies immobilized in the inner surface of microfluidic chips or beads coated with antibodies,80,81.Overall, microfluid-based isolating device on-a-chip provides convenience, the lab-on-a chip format can also be exploited to integrate multiple processes in a single instrument, simplifying operation and reducing the risk of cross-contamination. Moreover micro- and nano-scale devices exhibit high accuracy, precise control, lower energy consumption, cost reduction with respect to benchtop instruments and minimal sample size. A general disadvantage of all flow-induced methods is that they are based on microfluidics rules; hence, an exhaustive physical knowledge is needed for the design and operation of these devices. These microfluidic-based exosome isolation on-a-chip techniques have been reviewed in more detail in recent reviews. A number of conventional RNA extraction techniques are available, including phenol based techniques, combined phenol and column based approaches and pure column based methods. Eldh et al.[. They tested seven different methods: Trizol®, RNeasy®, miRNeasy, mirVana™, RNeasy®, modified RNeasy® and miRCURY™. All tested methods were able to extract RNA with high quality but with huge variation in the yield and size distribution of exosomal RNA. miRCURY™ showed the highest total RNA yield while Trizol®, miRNeasy and mirVana™ showed the lowest total RNA yield in exosomes. Moreover, the relative presence of smaller and larger RNA molecules was dependent on the used method. In short, column based RNA extraction techniques could extract RNA with a broad size distribution, whereas the pure phenol and the combined phenol-column extraction techniques seemed to be more efficient at extracting small RNA rather than total RNA. In terms of RNA purity Trizol®, modified RNeasy®, miRNeasy and mirVana™ showed a reduced purity comparing to the other strategies, probably due to residual phenol/guanidine. Since the different isolation methods give such an extensive variation in exosomal RNA yield and pattern, it is of main importance to select the RNA extraction method according to the aims of the research, especially in exosome research, since there is a relatively limited amount of exosomes from different body fluids.Once the exosomes have been isolated, the next step is RNA extraction. Exosomes are limited by a more rigid membrane compared to the cellular membrane due to the lipid composition, which would potentially affect the RNA extractiondh et al. performe. With the rapid development of next-generation sequencing (NGS) technology, the high-throughput sequencing has been widely used for discovery of candidate genes and lncRNAs. Similarly, lncRNA microarray is used to analyze lncRNAs differentially expressed. However, the cost of NGS and microarray analysis is relatively high, and the high amounts of data generated do not allow their use in clinical practice. Thus, nowadays the quantitative reverse transcription polymerase chain reaction (RT-qPCR) represents the most common detection method for detecting lncRNA with a relatively simple procedure.Finally, for the detection of lncRNA, after exosomal RNA isolation, several methods are in continuously innovation and improvement ,88. Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence. Some advantages of using northern blotting include: relatively fast, low -tech and cheap procedure; ability to detect the size of RNA fragment; observation of alternative splicing products; measurement of both RNA quality and quantity. Northern blot is a high specific method that reduces false positives and gives the opportunity to store membranes that can be reprobed years after blotting. Northern blot also has some pitfalls like: high risk of sample degradation by RNases; its low sensitivity and the use of some chemicals, such as formaldehyde, radioactive material or ethidium bromide used which can have harmful effects on health.In the following paragraphs, we will review the principles of the different methodologies for lncRNA detection as well as their pros and cons. Northern blot was more used in the past, however there are still some reports using this technique for lncRNA detection,36,91, which allows the quantification of lncRNAs expression in the sample thanks to transcript amplification and further fluorescent signal detection. It is a cost-effective and time efficient method, moreover it shows a high sensitivity and specificity, low amount of starting RNA is required, and the results are fast and easy to obtain, since it is not required any post PCR processing or data processing. However RT-qPCR has also some limitations such as: the amplicon size cannot be detected; maximum of four lncRNAs can be detected simultaneously; and non-specific binding can occur if SYBR® green is used, among others. Another technique commonly used to identify lncRNAs are microarrays,95, which consist of a library of DNA specific sequences (known as probes) immobilized in a grid. The isolated mRNA is reversed transcribed into cDNA incorporating fluorescent probes and hybridized to the microarray. Only the cDNAs complimentary to the probes, and thus the ones that are expressed, are detected by the laser scanner. This technology provides data for multiple lncRNAs in the same experiment, the protocols are well defined and standardized, and it is a relatively low-cost method. But it has also some disadvantages like: the analysis is only possible for known lncRNAs; the hybridization can be non-specific, thus giving false positives; high variability for low expressed genes; and not identification of lncRNAs sequence variants. RNA-seq is probably the most accurate technique to reveal the presence and quantify lncRNA in a biological sample at a given time. The fact that RNA-seq is not dependent on previous sequence information is a major strength, it has a high dynamic range and several isoforms of lncRNAs can be detected. Disadvantages are more related to the high cost of the technology and complex analysis process that requires high expertise of researchers. Interestingly Lane et al. recently published a book with extended information about methodologies for exosomal isolation and identification.The most used methodology for lncRNA is RT-qPCR. Several studies validated the utility of exosomal lncRNAs as minimally invasive diagnostic and prognostic marker in all kind of body fluids The continuous development of diagnostic techniques provides a technical basis for liquid biopsy in patients with cancer and for quantitative detection of circulating exosomal lncRNAs. By comparing the expression of lncRNA between normal population and cancer patients, the exosomal lncRNA-based liquid biopsy may be used as a diagnostic techniqueet al. analyzed the utility of the exosomal colorectal neoplasia differentially expressed-h (CRNDE-h) non-coding RNA in a cohort of 148 patients suffering from a variety of colorectal cancer (CRC) compared to control volunteers. They isolated RNA from serum-purified exosomes and measured the CRNDE-h expression levels by RT-qPCR. Their main findings indicated a significant correlation of the exosomal CRNDE-h lncRNA levels with the CRC regional lymph node/distant metastatic status. Moreover, ROC analysis showed the possibility to distinguish CRC patients, colorectal benign diseases and healthy individuals with 70.3% sensitivity and 94.4% specificity, which was superior to the traditional CEA tumor marker. In conclusion, detection of exosomal CRNDE-h lncRNA has a high potential as a noninvasive serum-based tumor marker for diagnosis and prognosis of CRC, for developing new therapeutic strategies. The survival of CRC patients is closely related to the stage at diagnosis; therefore, there is an urgent need for early diagnosis for CRC prevention and successful treatment. The study performed by Hu et al. aimed to identify potential exosomal lncRNAs that may use as early stage biomarkers for CRC. Microarray studies identified a subset of 6 lncRNAs  significantly higher expressed in the plasma of CRC patients than in negative control individuals. They isolated exosomal RNA from the plasma of 50 CRC patients and 50 healthy donors for validation of these potential biomarkers by RT-qPCR. The authors found a correlation between the high levels of expression of these markers and the presence of CRC. Furthermore, they analyzed the expression patterns in each stage of CRC and found the 6 lncRNAs significantly upregulated in stage I/II of CRC disease. These authors concluded that the 6 lncRNA panel can be used as biomarkers for non-invasive screening of CRC with high sensitivity and specificity. Li et al. also carried out an study to determine the usefulness of the exosomal GAS5 lncRNA (Exo-GAS5) as a biomarker for early stage non-small cell lung cancer (NSCLC) diagnosis. They analyzed by RT-qPCR the expression of Exo-GAS5 isolated in the serum from 64 NSCLC patients and 40 healthy donors, and found a lower expression in the former group. Moreover, those patients in early stage presented higher Exo-GAS5 levels compared with advanced-stage patients. The authors also demonstrated a better performance of the Exo-GAS5 than the conventional tumor marker CEA to distinguish NSCLC patients at early stages from healthy volunteers. Finally, they identified a significant association between lower Exo-GAS5 expression with larger tumor size and advanced TNM stage.Liu . However, this imaging technique is unable to distinguish between true progression from pseudo-tumor progression, which may have consequences in clinical decisions. Tan et al. designed a study including 43 GBM patients and 40 healthy donors to determine the feasibility of using exosomal HOTAIR lncRNA as a serum biomarker in conjunction with MRI to distinguish pseudo-progression and true progression. They assessed the HOTAIR expression levels by RT-qPCR and observed an increase in HOTAIR both in whole serum and purified exosomes but not in serum supernatant depleted of exosomes of GBM patients in contrast to control group. Moreover, they confirmed this finding by single molecular sequencing (SMS) analysis. Therefore, they proposed the detection of exosomal HOTAIR lncRNA in the serum of GBM patients as a noninvasive biomarker to detect GBM growth or recurrence. Similarly Tang et al. also showed the correlation of high serum exosomal HOTAIR levels with poor survival and poor response to chemotherapy in breast cancer patients. Briefly, they compared serum exosomal HOTAIR expression levels between 15 breast cancer patients and 15 healthy individuals, demonstrating a significantly higher expression in breast cancer patients than in healthy controls. They observed a marked decrease of serum exosomal HOTAIR lncRNA levels after 3 months of surgery and ROC analyses showed that serum exosomal HOTAIR had a significant greater capacity than the CA 15-3 antigen as a diagnostic and prognostic biomarker. In addition, results for the survival analysis revealed that high expression of exosomal HOTAIR lncRNA led to a worse disease-free survival and overall survival than low expression. Finally, the analysis of exosomal HOTAIR lncRNA expression in patients before and after treatment suggested that high levels may lead to a poor response to neoadjuvant chemotherapy and to tamoxifen hormone therapy.Glioblastoma multiforme (GBM) is the most common and aggressive malignant adult primary brain tumor. After tumor resection, patients are monitored by magnetic resonance imaging (MRI)et al. validated a panel of three lncRNAs  by RT-qPCR, in urine exosomes (UE) of 80 bladder cancer patients and 80 healthy controls, and found a significant overexpression in bladder cancer patients. The verification of the diagnostic capacity indicated a much better performance for this panel than for the urine cytology, which is widely used in clinical practice. Moreover, they identified a significant correlation between the overexpression of UE-derived PCAT-1 and SPRY4-IT1 and the advanced TNM stage. The utility of UE as a liquid biopsy in bladder cancer was confirmed by Yazarlou et al.. The study included a total of 59 BC patients, 24 healthy individuals and 25 patients with non-malignant urinary related disorders. They evaluated expression of several lncRNAs in urinary isolated exosomes by RT-qPCR in all the study participants. They found a significantly higher expression of LINC00355, UCA1-203, and MALAT1 and a decreased UCA1-201 expression in bladder cancer patients compared to the other two groups. Among all these lncRNAs, UCA1-201 transcript levels had the best performance to discriminate between bladder cancer from normal/nonmalignant disease samples, and thus indicating its potential use as a prognostic biomarker in BC.Exosomal lncRNAs can be also isolated from other body fluids rather than the most common, such as plasma or serum. For instance, Zhan et al. detected by RT-qPCR the presence of the MALAT-1 lncRNA in the saliva of all oral squamous carcinoma patients analyzed (n = 20), despite mRNA expression was not significantly different between metastatic and non-metastatic samples. However, HOTAIR was detected mostly in patients with lymph node metastasis, compared with those without metastasis. These results indicated a promising use lncRNAs in saliva as a noninvasive and rapid diagnostic tool for the diagnosis of oral cancer, but a higher number of samples must be included to confirm that results.Saliva is another source of lncRNA and liquid biopsy that has been proposed for clinical application. Tang . Zhang et al. analyzed the expression level of exosomal RP11-838N2.4 lncRNA by RT-qPCR in 78 serum samples from patients with advanced NSCLC receiving erlotinib treatment. They identified the involvement of the exosomal RP11-838N2.4 lncRNA in the modulation of chemotherapeutic responses, suggesting its potential role as a novel target for prediction of NSCLC treatment with erlotinib.In addition to their value as diagnosis and prognosis biomarkers in cancer, exosomal lncRNAs were involved in drug resistance of tumor cells, suggesting a clinical application in cancer-targeted therapy. A recent study demonstrated the therapeutic benefit of erlotinib in patients suffering from NSCLC in large randomized phase III studies, however the majority of these patients exhibited erlotinib-refractory disease, acquiring chemoresistance. In this context, there is an urgent need to elucidate the mechanisms of erlotinib resistance and to discover reliable biological targets that play important role in erlotinib resistance. Several in vitro assays performed by Dong et al. confirmed that the exosomal SNHG14 lncRNA was essential for trastuzumab resistance in breast cancer. They validated this statement in serum samples from a cohort of 72 patients with advanced HER2+ breast cancer treated with trastuzumab. Their results showed an increased serum exosomal SNHG14 lncRNA expression level, determined by RT-qPCR, in patients who did not respond to the treatment compared to good responders. ROC analysis demonstrated a high sensitivity and specificity of SNHG14 lncRNA as a potential diagnostic biomarker for trastuzumab breast cancer treatment. Moreover, they also observed an increased expression of exosomal SNHG14 lncRNA was markedly associated with distant metastasis, lymph node metastasis and cardiac toxicity.Trastuzumab is effective used for early stage treatment of breast cancer patients bearing positive HER2 mutations, however, after a period of exposure, some patients acquire resistance. Then, there is a clear need for useful therapeutic biomarkers able to predict chemoresponses to treatment with trastuzumab. Sunitinib, which has potent anti-angiogenic effects and direct antitumor activities, was proposed as an effective therapy for RCC patients. However, the development of sunitinib resistance was observed resulting in failure of sunitinib and reduced survival rate. The in vitro studies carried out by Qu et al.[n = 74). Additional analysis let to conclude that high lncARSR levels in plasma correlated with poor sunitinib response in RCC patients. They investigated further into the mechanism of resistance to sunitinib indicating that it was required lncARSR to be packed in exosomes to exert resistance. Moreover, they observed that lncARSR incorporated into exosomes and transmitted to sensitive cells, resulted in sunitinib resistant cells, thereby disseminating drug resistance. All together the authors demonstrated not only potential utility of exosomal lncARSR as a clinical biomarker to sunitinib response but also as a therapeutic target to overcome sunitinib resistance and improve the clinical benefits in RCC patients. Thus, liquid biopsy by detecting exosomal lncRNA provides a minimally invasive approach, a real-time monitoring of drug response, and accurate information for clinicians to administer a reasonable medication.In renal cell carcinoma (RCC) nearly 30% of patients develop recurrence and metastasis after tumor resectionQu et al. pointed , and exosomal lncRNAs can be stably detected in human body fluids because of the protection of exosomes. This together with the continuous development of lncRNA detection techniques has promoted exosomal lncRNA-derived liquid biopsy to become a novel approach in the diagnosis, prognosis, and treatment of cancers. As described in the section 4 of this review the studies on exosomal lncRNAs are accumulating, however its clinical use is not really extended yet. Indeed, liquid biopsies are not currently considered as a standard operating procedure for the diagnosis of cancers and for the characterization of the disease. Instead it is used as a complimentary test to tissue biopsy. However, cancer patients are often unable to undergo highly invasive examinations because of their poor physical condition, thus a minimally invasive, reproducible, real-time detection is extremely desirable. Despite the numerous studies in liquid biopsy, clinical validations are mandatory to assess its accuracy explaining why its use is not very widespread in medical practices.LncRNAs were confirmed to be closely related to the development of cancerTo use exosomal lncRNAs as novel biomarkers, high purity and accuracy techniques for their isolation and detection are required. Currently, many methods were developed for and each of them has many advantages and disadvantages which must be evaluated. The method used should be selected according the objectives of the study., affecting RNA liquid biopsy performance. Thus, the exact contribution of these variables must be rigorously assessed. A recent initiative for this purpose is “The extracellular RNA quality control (exrnaqc) study” carried out by Decock et al.. The most appropriate source  of exosomal lncRNAs must be defined. For instance, results from lncRNAs extracted from whole blood should be interpreted cautiously due to lncRNAs contained in blood cells and/or circulating cancer cells and/or systemic inflammatory context that could result in important deviations. The individual variability/heterogeneity in lncRNA tumor expression patterns (intra- and inter-tumor heterogeneity) should be considered as well as other external variables such as diet, physical activity or drugs,116. Technological improvements of lncRNA measurement are then needed to improve its sensitivity, normalization as well as data analysis. For instance, total RNA sequencing methods are still difficult to implement in many research laboratories and would benefit from further technological improvement. Notably, in the vast majority of the studies that we found in the literature, the detection of lncRNA is currently carried out by RT-qPCR, however the cost related to the yield is still too high. A similar emerging technology is digital PCR (dPCR) characterized by a highest robustness and technical reproducibility as well as an improved sensitivity (30-fold) and accuracy (10-fold). Therefore, dPCR represents a very promising method for detecting lncRNAs in liquid biopsies with low nucleic acid content. Recent studies already reported the use of dPCR to detect exosomal lncRNAs,120. However, data normalization is required to reduce the variation associated with sample collection, processing and measurement. Despite of some studies on exosomal lncRNAs showed a better performance than the conventional biomarkers in terms of prognosis and diagnosis,101, further investigations are necessary to confirm if lncRNAs can be used as independent biomarkers or should be used in combination with the existing ones. Multicentric clinical studies will be also required before to use lncRNAs as diagnostic biomarkers.Many challenges regarding exosomal lncRNAs need to be confronted. For instance, physiological and pathological roles of exosomes and exosomal lncRNAs in the tumor microenvironment remain to be further explored. In addition, the heterogeneity of exosomes in body fluids may be a drawback to their use as biomarkers, since can lead to false negatives or positives in cancer diagnosis. Prior to the application in the clinical context, standardized methods of preparation of exosomal lncRNAs from biofluids are mandatory. Indeed, many pre-analytical factors such as time of fluid collection, fluid preservation or RNA isolation protocol among others, influence RNA abundance levels in body fluids both inside and outside extracellular vesiclesThe recent discovery of the association of exosomal lncRNAs and cancer progression has attracted the attention of researchers and opened up exciting prospects for diagnostics, prognostics and drug response monitoring. Elucidating the exact roles of exosomal lncRNAs in tumorigenesis, metastasis, and drug resistance, as well as the development and application of standard operating procedures, are areas of considerable interest to offer a more sensitive and accurate biomarker for early cancer detection or to improve the efficiency of existing clinical biomarkers.


Increasing physical activity (PA) in rural communities is a vital prevention tactic in multiple chronic diseases; however, little is known on the multilevel correlates of PA rural areas. A better understanding of domain-specific PA adds context for promoting PA in rural communities. The current study sought to determine factors associated with domain-specific and overall moderate-vigorous physical activity (MVPA) in rural communities.M = 57.0 [SD = 16.7], 67.8% female, 83.8% white). Generalized linear models with negative binomial distributions examined the relation between demographics, trail use, and perceptions of the neighborhood environment, with domain-specific and overall MVPA, measured via the Global Physical Activity Questionnaire.Surveys were conducted across 14 rural mid-Western communities, with the final analytical sample including 1241 adults , with 58.5% meeting MVPA guidelines. Higher age, female gender, and higher educated individuals had lower levels of overall and occupational MVPA. Females, higher education, and perceived indoor recreational access were associated with lower levels of transportation-related MVPA, while trail use was associated with increased transportation MVPA. Higher age and female gender respondents had lower levels of recreational MVPA, while trail users and those who perceived favorable indoor recreational access had higher levels of recreational MVPA.PA primarily occurred in the occupational domain among this sample of rural mid-Western adults. Findings highlight the need for multilevel interventions to address PA across multiple domains in rural communities, especially among females and older adults. Physical activity (PA) has myriad benefits including improved weight status, reduced risk of various cancers and cardiovascular disease . Most AmPhysical activity is a complex behavior with a multitude of influences, and is strongly influenced by environmental and policy influences. Multilevel factors encourage engagement in PA such as the environment  and socIn 2016, a specific call to action for rural active living promoted the application of a refined ecological approach to determine the correlates of domain-specific PA in rural communities . FurtherIn another study among rural Midwestern adults, unique correlates were found for the previously discussed domains of PA and other domains such as active transportation, yard work in conjunction with housework, and recreation . The resThe literature is rich in evidence supporting the impact of individual and environmental level correlates of recreational and overall PA, yet lacks in providing evidence in the context of rural communities. Furthermore, the understanding of individual and environmental level correlates of domain-specific PA in rural communities remains limited. Therefore, to better understand the correlates of overall and domain-specific  MVPA in rural communities in order to inform PA interventions, the objectives of the present study were to (1) provide estimates of overall and domain-specific total weekly MVPA, and (2) examine associations of individual and environmental factors with overall and domain-specific weekly MVPA minutes.The current study used baseline data collected from August 2019 to September 2020 from a sample of rural community members participating in a physical activity intervention across a rural Midwestern region. Adult participants who were able to be physically active were recruited by address-based sampling, referral, and community outreach across 14 rural communities in the region – more detailed information on sampling and study eligibility can be found elsewhere . Prior tPhysical activity (PA) was assessed using the Global Physical Activity Questionnaire (GPAQ) , 19. TheParticipants provided demographic information including age, gender, education, race/ethnicity, and income. Age was operationalized according to the PA Guidelines for Americans . Gender and education were dichotomized into male or female and high school degree or less and greater than a high school degree. Race was also dichotomized into white or non-white and income of less than $50,000 or greater than $50,000 via median split, respectively. Individual characteristics related to physical activity behavioral factors were also collected and included trail use. Trail use was characterized by participant’s reporting having used their local trail or not.Three perceived neighborhood environment subscales were used from the abbreviated Neighborhood Environment Walkability Scale (NEWS), along with the Rural Active Living Perceived Environment Support Scale (RALPESS) , 21. TheDescriptive statistics including means and frequencies were captured for all variables of interest . To account for missing data from variables of interest, multiple imputation was performed using multivariate imputation by chained equations . AdditioThe analytical sample dropped from 1252 participants to 1241 participants due to relevant missing data. The sample  was predominantly female and white  and environmental  correlates were statistically significant Table . When coWhen examining individual and environmental correlates of weekly recreational MVPA minutes, both individual  and environmental  correlates were statistically significant Table . For oldThis study was among the first to elucidate multilevel correlates of overall and domain-specific PA in a rural population. The findings of the study demonstrate the influence of age, gender, race, and education on overall and domain-specific PA. Further, this sample of rural residents had high levels of occupational PA and conversely, low levels of transportation PA. Findings from this study contribute to the gap in literature regarding multilevel correlates of domain-specific PA in rural communities, and will benefit the design and adaptation of interventions seeking to improve levels of domain-specific PA, ultimately reducing adverse health outcomes, among rural community members.Consistently across the domains and overall PA, increased age was found to be associated with decreasing PA. PA is well known to decrease as age increases across multiple domains . The traIn addition to being influenced by age, overall MVPA and every MVPA domain were associated with various individual level correlates. In particular, the gender of a participant determined the extent to which a participant engaged in overall MVPA and each MVPA domain. The trend maintained consistency across the different MVPA outcomes, where female participants engaged in significantly fewer weekly minutes of overall MVPA, occupational MVPA, transportation MVPA, and recreational MVPA than male participants. In general, women are known to be less physically active than their male counterparts in rural areas , 16, 28.Our findings highlight a significant difference in weekly minutes of occupational and transportation MVPA in addition to overall and recreational MVPA based on gender. Females in this sample of rural communities engaged in fewer minutes of weekly recreational MVPA than males, a finding aligning with previous conclusions of gender differences in the recreational MVPA domain in rural communities . Unlike Educational attainment and PA have historically shown a direct relationship , 30–32; Lastly, trail use and perceived indoor recreation access were predictive of weekly minutes of recreational and transportation MVPA among rural community members. More walking trail use has been associated with more weekly total PA ; howeverThe cross-sectional nature as well as the self-reported variables limit the ability to distinguish causality. This study took part in a specific geographic region of the U.S. thereby limiting the generalizability of the findings. Not all levels of the socioeconomic model  were measured and analyzed in this study. Future analyses should include the measurement and subsequent analysis of other important covariates. There was a lack of diversity in the study sample; however, the sample demographics were aligned with regional demographics. Lastly, the PA data was self-reported, which can be overestimated.Despite the limitations, this study has several strengths. The study attained a relatively large sample size containing respondents of the baseline survey, especially considering the sample consisted of rural community members. The fact that the study focused analyses on participants from rural communities in particular represents another strength, due to the frequent under-representation of the rural demographic in PA research and in human subjects research in general. Lastly, the study revealed preliminary evidence for the individual and environmental correlates of not just overall PA, but also domain-specific PA in rural communities to identify potential targets for future interventions seeking to improve PA in rural communities.It is imperative, when measuring PA, to include the domain of the activity along with a broad array of contextual variables. Commonly, PA is viewed as an overall concept where various domains of PA are summed to create a dichotomous variable of meeting guidelines; however, actionable information may be lost when coalescing PA across domains. Physical activity comes in many forms and this study found occupational and recreational PA as important domains for rural people. By examining multilevel correlates of specific domains of PA, we can have a deeper understanding on how to best promote physical activity for different groups in different environments. Ultimately, more targeted interventions could help increase PA and reduce chronic disease at the population level in rural communities.


Sinus of Valsalva aneurysm is a rare cardiac abnormality which can be acquired orof congenital origin. A spontaneous rupture into the right atrium is possibleand, if not adequately treated, may result in a progressive heart failure due tothe left-to-right intracardiac shunt. If ruptured sinus of Valsalva aneurysm isdiagnosed, surgical repair is indicated, and different surgical techniques havebeen reported. If concomitant aortic regurgitation is present, aortic valvereplacement is usually performed. Herein, we describe an uncommon clinicalpresentation of a ruptured sinus of Valsalva aneurysm which has been correctedby aortic valve reimplantation. Most commonly, the aneurysm islocated either in the right or the non-coronary sinus while the left coronary sinusis rarely involved. During thetime course, a spontaneous rupture of sinus of Valsalva aneurysm into the rightatrium is possible and, if not adequately treated, may result in a progressive heartfailure due to the left-to-right intracardiac shunt or even in a sudden cardiacdeath. Depending on thesize of perforation, clinical presentation may vary between an asymptomatic heartmurmur, mild dyspnea, chest pain, or even signs of acute heart failure. Diagnosis is confirmed with transesophagealechocardiography (TEE), contrast-enhanced computed tomography (CT), or magneticresonance imaging. If rupturedsinus of Valsalva aneurysm is diagnosed, surgical repair is indicated and differentsurgical techniques have been reported, depending on the type of theaneurysm. If concomitantaortic regurgitation is present, aortic valve replacement is usuallyperformed. Herein, wedescribe an uncommon clinical presentation of a ruptured sinus of Valsalva aneurysmwhich has been corrected by means of aortic valve reimplantation and simultaneousaortic valve repair.Sinus of Valsalva aneurysm is a rare cardiac abnormality which can be of congenitalorigin (TM Valsalva Graft to mimic the original valveorientation and to achieve sufficient coaptation of aortic cusps. In addition,central plication suture of the right coronary cusp was performed to reach theeffective cusp height of 8 mm. Furthermore, pulmonary vein isolation was performedwith bipolar radiofrequency and the left atrial appendage was closed using anAtriClip® LAA Exclusion System. After weaning from cardiopulmonary bypass,the TEE showed a normal aortic valve function with only trace residual aorticregurgitation, no residual left-to-right cardiac shunt, and complete closure of theleft atrial appendage. The patient recovered uneventfully after the surgery and wasdischarged in stable sinus rhythm and only trace aortic regurgitation with a meantransvalvular gradient of 13 mmHg.A 57-year-old male patient was referred to our hospital for catheter-based ablationdue to paroxysmal atrial fibrillation. The patient reported intermittentpalpitations, dizziness, and a reduced quality of life. Otherwise, the patient washealthy and had no previously diagnosed connective tissue disorder. During thepreprocedural diagnostic work-up, a TEE was performed and revealed an ascendingaortic aneurysm of 43 mm and a concomitant sinus of Valsalva aneurysm of 20 ×20 mm protruding into the right atrium  associatThis case represents an aortic valve sparing root procedure which aims to maintainthe geometry of aortic valve considering the marked asymmetry of aortic root. Thechallenge of reimplantation procedure in this scenario is to keep the asymmetricconfiguration of aortic root due to extremely dilated non-coronary sinus and largenon-coronary cusp of the aortic valve. Aortic valve annulus was almost non-existentbelow the perforated sinus of Valsalva aneurysm in the non-coronary sinus and wastherefore fixed from inside to outside using the standard pledgeted subannularanchoring sutures. In this specific case, the defect was in the right atrial groovewell above the tricuspid valve plane. Therefore, the right atrium was not separatelyopened.Despite an uncommon clinical presentation, a ruptured sinus of Valsalva aneurysm canbe detected by comprehensive preoperative work-up. Further, an aortic valvereimplantation is possible even in a complex asymmetric anatomy if the geometry ofthe aortic valve is respected.


Contingency management is highly effective but highly underutilized.Low uptake is largely attributed to a lack of innovation and moral, ethical, and economic concerns, among other barriers.Technology-enabled solutions and academic–industry partnerships are critical to advance opioid use disorder care.Contingency management  is backed by decades of empirical support yet remains highly underutilized. Rewards are rarely used in real-world clinical practice due to a number of concerns, including most notably, the apparent lack of innovation, as well as moral, philosophical, ethical, and economic concerns, and even federal rules meant to prevent illegal inducements in health care. Still, other opponents argue that some patients will try to "game" the system by simply doing whatever it takes to earn monetary rewards. This paper provides a succinct, up-to-date overview of the current evidence base for contingency management for opioid use disorder. Common barriers and solutions to implementation, as well as implications for future research and clinical practice are discussed. Although important, greater uptake of contingency management interventions is about more than legislation and regulations; it’s about recognizing stigma, shaping attitudes, and increasing awareness. Provider involvement in advocacy efforts at all levels and collaboration involving academic–industry partnerships is necessary to advance the burgeoning digital health care space and improve outcomes for people with opioid use disorder. America’s drug overdose crisis—largely driven by opioids—is arguably one of the greatest public health concerns of our time with significant loss of life and economic burden. Over100,000 Americans died of a drug overdose in the past year with roughly 75% attributed to opioids ,3. AmeriGiven the chronic, often relapsing course of severe OUD, effective treatment requires long-term management. Medication treatment with methadone or buprenorphine is a viable treatment option associated with a variety of positive outcomes, including most notably, reduced risk for all-cause and overdose mortality . Poor adCohen d = 0.70) on abstinence from stimulants among patients receiving medication for OUD [When combined with medication for OUD, contingency management demonstrates significant reductions in morbidity and mortality from OUD, considerable cost savings, and reduced hospitalizations and emergency department visits ,20. Cont for OUD , which i for OUD ,21,22. TDespite decades of research supporting the effectiveness of contingency management and generally positive beliefs held by front-line addiction treatment providers and patients alike ,24, its A leading barrier to greater adoption of contingency management in clinical practice is presumably the relative lack of innovation. The origins of contingency management date back to the 1960s and operant conditioning principles, which was followed by a surge of rigorous, high-quality clinical trials in the 1990s. Traditional contingency management protocols  have since become rudimentary, outdated, and onerous in the current digital era, necessitating novel, technology-enabled solutions to facilitate widespread adoption. Other limitations include that many accepted contingency management procedures reward drug-free urinalysis screens exclusively, and there is only a low chance that the desired behaviour will actually be reinforced. In the commonly used probabilistic “prize-based” procedure, patients earn draws from a prize bowl containing slips of paper when the target behaviour is exhibited, but slips often have either no monetary value or a low-value prize. This raises the common complaint that contingency management is a “game of chance” due to the lack of immediate and consistent meaningful reinforcement that is required for lasting change.There has been rapid progress on the innovation front in recent years with respect to emerging technologies leveraging contingency management in the treatment of OUD ,28,29. HA number of studies  have ideOther opponents may argue that addiction treatment patients will try to "game" the system by simply doing whatever it takes to earn monetary rewards, thereby questioning patients’ true motivation “to get better.” When hearing this tired argument about people with addiction, I often find it helpful to consider patients diagnosed with a different chronic medical condition. Consider, for example, a patient with hypertension, diabetes, or obesity who earns $1 for each day they engage in 30 min of exercise, solely motivated by the modest financial incentive. Would this patient not still experience positive health benefits over time  irrespective of their underlying motivation? Many health plans already offer incentives to their members who engage in various healthy lifestyle behaviours . What if we rewarded OUD patients with incentives for not only producing negative urinalysis drug screens, which is often the exclusive target behaviour of traditional contingency management protocols, but also engaging in recovery-oriented behaviours such as attending therapy sessions, taking their medication , and participating in community-based mutual-help support groups? In fact, this broadened approach to contingency management rewards has been used to good effect in several studies ,43, and via mobile app on medical costs due to hospital-based encounters and procedures among a sample of OUD patients treated with buprenorphine documented that the medical cost reduction in patients using the app relative to those receiving standard care offset the cost of the digital therapeutic itself, thereby resulting in a net cost-savings of $720 per patient [Program administrators, policy-makers, and payers may understandably voice concerns about increased treatment costs associated with providing monetary incentives given that contingency management is often an “add-on” to usual care (i.e. adjunctive intervention). However, contingency management, when combined with medication treatment for OUD, has demonstrated the largest cost-savings relative to other evidence-based interventions for OUD, including medication alone . A recen patient .The cost-saving benefits of reduced hospitalizations and emergency department visits for patients receiving contingency management, while certainly viewed as a positive outcome by treatment staff directly involved in the patient’s care, are often only realized by payers. Although many effective contingency management interventions are limited to around $100 a month in rewards , in the via federally-funded health plans or programs [Also of interest are legal concerns and whether the use of monetary incentives violates federal and state law because it could be considered unlawful to give patients money who are enrolled in federally- or state-funded health plans or programs. The federal anti-kickback statute provides for criminal penalties for providers who knowingly and wilfully offer, pay, solicit, or receive remuneration to induce or reward, among other things, the referral of business reimbursable under any of the federal health care programs (Medicare and Medicaid). When incentives exceed nominal monetary values, they can be considered kickbacks or inducements per federal and state laws intended to prevent fraud, waste, and abuse. Several leading professional organizations and advocacy groups  have called on the Office of Inspector General (OIG) to create a new safe harbour provision protecting the use of payments provided as part of contingency management for patients receiving treatment programs ,48. EstaThe Office of Inspector General (OIG) published a final rule in December 2020 amending safe harbours to the federal anti-kickback statute . AlthougThe Biden-Harris administration has been transparent in its support for expanding access to evidence-based treatment, including contingency management . The Offvia multiple easy and convenient methods. Supplementing patient or collateral self-report, smartphone video and GPS location capabilities, as well as external testing hardware have all been used to good effect to monitor and confirm medication adherence, abstinence, and appointment attendance [Barriers to widespread adoption of contingency management in routine clinical practice, although pervasive, can be overcome. A number of innovative technologies, as described below, now allow for many aspects of contingency management to be fully or partially automated, thereby addressing common logistical barriers to implementation. Patient-facing mobile apps, combined with provider-facing dashboards, can facilitate tracking progress towards recovery goals and overall program-level management of the selected rewards system. Full automation is any solution not requiring action or verification by treatment staff before rewards can be delivered, whereas partial automation involves rapid delivery of rewards for certain recovery-oriented behaviours with other behaviours requiring verification by individual providers. Depending on the identified target behaviours, validation can be achieved tendance ,51–54. RAutomated delivery of monetary rewards can be achieved by linking a contingency management mobile app to a pre-paid debit card with the option to apply spending restrictions. “Smart” debit cards allow card administrators (e.g. treatment program staff) to toggle specified blocking capabilities on/off to prevent cash withdrawals or purchases at identified high-risk vendors . Both providers as well as patients in OUD treatment settings overwhelmingly prefer the use of “smart” pre-paid debit cards relative to giving patients actual cash, and view spending restrictions as an appropriate safeguard, particularly early on in one’s recovery . ClinicaEducation and training may be indicated to improve uptake of contingency management by addressing perceived skill deficits and competence needs of clinical staff ,56, who Involvement in local, state, and federal advocacy efforts to make contingency management more mainstream is required to lessen treatment providers’ perceived exposure to risk in providing payments to patients as part of contingency management interventions. At the federal level, this involves continuing to push for a safe harbour provision protecting the use of motivational incentives for patients enrolled in federally-funded health plans or programs, and participating in public comment periods. A newly created safe harbour specifically covering contingency management would ensure that despite potentially implicating the federal anti-kickback statute, contingency management would not be treated as an offence under the statute. Addiction treatment professionals, including executives, clinical directors, and front-line clinicians, are encouraged to familiarize themselves with their local and state elected officials, as well as their own state’s statutes regulating contingency management and the use of motivational incentives for state-funded programs as a logical first step, and then taking action, if necessary. Following local, state, and federal legislators on social media (as well as leading addiction policy advocates and organizations) also allows the opportunity to keep abreast of the latest relevant policy-related issues impacting addiction treatment and recovery. Greater acceptance of contingency management involves not only fixing legislation and regulations preventing uptake, but recognizing stigma, shaping attitudes, and increasing awareness. In addition to legislative advocacy, clinical staff can become a local champion and change agent for contingency management at their treatment program or health care system by proposing in-service trainings and calling out stigma when present by patients, colleagues, or staff. They can use their voice to advocate for compassionate, common-sense approaches and expand access to evidence-based interventions like contingency management for people with OUD. Educating others, raising awareness, and holding policy-makers accountable are small but effective ways to have a measurable impact on uptake and adoption of contingency management.Academic-industry partnerships are critical to building, testing, and scaling technology-enabled, reward-based solutions for OUD. Such collaborations could substantially advance addiction treatment systems by jointly bringing intuitive, science-backed solutions to market, which serve to improve uptake of contingency management and increase the availability of treatment slots by leveraging the efforts of program staff, particularly in underserved areas, where staff shortages are common. Although there is great potential for technology-enabled solutions to further support the prevention, treatment, and recovery of OUD by addressing practical barriers to adoption, OUD mobile apps often lack empirical evidence from well-designed studies supporting their use . As a nuThe U.S. digital health market is in the midst of considerable growth with total venture capital invested through mid-2021 at nearly $15 billion , surpassThe benefits of academic-industry partnerships flow both ways. Clinical researchers stand to gain just as much from the business know-how, technological expertise, marketing, and access to alternative and more streamlined lines of funding afforded to them by partnering with a digital health company. Moving beyond siloed academic work environments by forming strategic partnerships with industry provides researchers a front row seat to the practical application of their scientific findings, opens doors that may not have been possible otherwise, and is conducive to innovation. Recognizing the value of academic-industry collaboration, NIDA’s Small Business Technology Transfer (STTR) Program offers grant funding ranging from $150 K for Phase I up to $3 M for Phase II to foster technology transfer through cooperative R&D between academic researchers from non-profit universities and for-profit small business startups. A true bi-directional approach has the potential to shape and inform scientific research questions with an eye towards real-world challenges to implementation, ultimately improving contingency management solutions.In order to further advance the extant knowledge base on contingency management in general and technology-enabled solutions in particular, there are a number of areas warranting further work. Efforts targeting barriers to more wide-scale acceptance and implementation of contingency management, including most notably the apparent lack of innovation, would benefit from further empirical studies identifying specific technology-related reasons cited by program administrators and front-line clinical staff for the low rates of adoption in real-world treatment settings. The overwhelmingly majority of contingency management research studies conducted to date come from the U.S., and even fewer studies have examined contingency management for OUD specifically outside of the U.S. ,67. A reIt is also critical that contingency management researchers continue to expand on existing work to explore the feasibility and effectiveness of rewarding additional recovery-oriented behaviours beyond negative urinalysis drug screens. Although there is some evidence suggesting that rewarding attendance at appointments is equally effective to rewarding abstinence with respect to treatment engagement ,43, furtContingency management must be used as a critical front-line treatment for OUD in conjunction with life-saving FDA-approved medications. Despite decades of rigorous clinical trials and robust meta-analyses showing considerable benefits for contingency management interventions, a number of barriers precluding widespread adoption persist. Fortunately, many barriers can be overcome by holding specialized staff trainings, actively calling out stigma, advocating locally and nationally, fostering academic-industry partnerships, and incorporating innovative technologies. Adoption of technology-enabled contingency management solutions, that reward more than simply negative drug screens to include additional recovery-oriented behaviours ,43, is a


Inhibin A and B are heterodimeric proteins of inhibin alpha (INHA) and beta (INHBA or INHBB) subunits, and homodimers of two beta subunits form activins. They belong to the transforming growth factor beta superfamily of pleiotropic signaling molecules. While inhibins are mainly formed in gonads, activins are widely produced in extragonadal sites. Their classical action is to regulate positively (activin) or negatively (inhibin) pituitary secretion of follicle-stimulating hormone (FSH), but activins also have diverse non-endocrine effects, including cell proliferation, inflammation and carcinogenesis . Both prBetaglycan, the receptor participating in inhibin’s inhibitory action on activin, is expressed in human testis in primary spermatocytes, round spermatids and in Leydig cells (LC), which are thus potential targets of intratesticular inhibin action . ActivinInha−/− mice develop infertility and gonadal sex cord stromal tumours, of SC origin in males, followed by a cachexia-like wasting syndrome  and FSH levels, low testosterone, highly elevated anti-Müllerian hormone (AMH) and very low to nondetectable inhibin B. Both men were found to carry a homozygous 2-bp deletion  in the INHA gene, resulting in frameshift of the message and truncation of the INHA protein. Although functional studies on the mutant gene were not carried out, it is likely it resulted in complete loss of inhibin production. Unfortunately, due to logistic problems, activin levels could not be measured. Circumstantial evidence for increased activin action at the pituitary levels was provided by the strongly elevated FSH levels. Testis biopsy was not carried out.Very little is still known about the consequences of genetic inhibin and activin inactivation in humans, apart from some reports on heterozygous mutations associated with premature ovarian failure and gonadal tumours , 8. The et al.  on two cThe phenotype of the men can be explained by current knowledge of inhibin and activin actions, mainly for animal models. Strong activin action has been shown to stimulate SC proliferation but inhibit their maturation . This maInha knockout mice, had no signs of testicular tumours or cachexia. It will be warranted to keep these conditions in mind upon follow-up of the patients because we do not know whether they represent a genuine species difference or may take longer to appear in humans. Long-term follow-up of the brothers will undoubtedly reveal us more about the effects of missing inhibin action. The histology of large testes devoid of complete spermatogenesis would be of particular interest, if testicular sperm extraction were to be used for infertility treatment. Human chorionic gonadotrophin treatment, in order to increase intratesticular testosterone levels, is unlikely to cure azoospermia, because of the activin-induced maturation arrest of SC. Attempts to suppress activin action would probably be more promising. Maybe the novel inhibitors of the activin receptor signaling pathway could offer a solution for reversing the azoospermia (The men, unlike ospermia .The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.


Breast cancer (BC) is the most frequently diagnosed female invasive cancer in Western countries and the leading cause of cancer-related death worldwide. Nowadays, tumor heterogeneity is a well-known characteristic of BC, since it includes several nosological entities characterized by diﬀerent morphologic features, clinical course and response to treatment. Thus, with the spread of molecular biology technologies and the growing knowledge of the biological processes underlying the development of BC, the importance of imaging biomarkers as non-invasive information about tissue hallmarks has progressively grown. To date, breast magnetic resonance imaging (MRI) is considered indispensable in breast imaging practice, with widely recognized indications such as BC screening in females at increased risk, locoregional staging and neoadjuvant therapy (NAT) monitoring. Moreover, breast MRI is increasingly used to assess not only the morphologic features of the pathological process but also to characterize individual phenotypes for targeted therapies, building on developments in genomics and molecular biology features. The aim of this review is to explore the role of breast multiparametric MRI in providing imaging biomarkers, leading to an improved differentiation of benign and malignant breast lesions and to a customized management of BC patients in monitoring and predicting response to treatment. Finally, we discuss how breast MRI biomarkers offer one of the most fertile ground for artificial intelligence (AI) applications. In the era of personalized medicine, with the development of omics-technologies, machine learning and big data, the role of imaging biomarkers is embracing new opportunities for BC diagnosis and treatment. Moreover, the introduction of established tumor biomarkers in the most recent edition of Tumor Node Metastasis (TNM) staging system by the American Joint Committee on Cancer for several tumor entities, including BC, illustrates the movement in progress towards precision approaches and therapies. Within the framework of precision medicine, biomarkers become an important element for developing study methodology, research hypotheses and selectively applying scientific findings in cancer care. Imaging findings were only recently officially recognized as biomarkers even if it is in the intrinsic nature of imaging to be applied in this sense. BC was the most frequent cancer diagnosed among females in 2020 and breast MRI has been established as a non-invasive imaging modality for the detection, characterization and local staging of breast tumors with several recommendations including screening of high-risk females, pre-operative local staging and systemic therapy monitoring. Contrast-enhanced MRI (CE-MRI), diffusion-weighted imaging (DWI) and magnetic resonance spectroscopy (MRS)-based imaging biomarkers have shown to be highly correlated with BC molecular subtypes and other prognostic and predictive factors. Furthermore, multiparametric MRI (Mp-MRI) approaches have been introduced to investigate associations of imaging biomarkers with histological types and subtypes, response to treatment, risk of recurrence and overall survival in BC patients.In recent years, with the spread of molecular biology technologies and the increasing knowledge about the biological processes underlying cancer development, considerable interest in biomarkers has progressively grown. In 2016, the latest glossary released by the U.S. Food and Drug Administration (FDA) - National Institutes of Health (NIH) Biomarker Working Group in its Biomarkers, Endpoints, and other Tools (BEST) Resource, defined a biomarker as “a defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes or responses to an exposure or intervention, including therapeutic interventions. Molecular, histologic, radiographic or physiologic characteristics are types of biomarkers”. Breast imaging biomarkers can be divided into qualitative, ordinal and quantitative as shown in  The American College of Radiology Breast Imaging-Reporting and Data System (ACR BI-RADS) lexicon is the first and the best validated system of imaging descriptors in radiology. Ordinal biomarkers are categories with intrinsic rankings that can be arranged in a meaningful order. Breast MRI background parenchymal enhancement (BPE) with minimal, mild, moderate and marked categories is an example of ordinal biomarkers. Quantitative biomarkers are objective, measurable and reproducible parameters. Anatomic structures 2D and 3D measurements are examples of quantitative biomarkers essential in diagnosis, staging and monitoring of response to treatment, as it is when applying the Response Evaluation Criteria in Solid Tumors (RECIST 1.1) criteria. Additional quantitative biomarkers derived from breast Mp-MRI, are apparent diffusion coefficient (ADC) maps values of DWI and the transfer constant (Ktrans) that provides a measure of capillary permeability with CE-MRI perfusion. The use of panels or scoring systems combining multiple imaging parameters, such as TNM, can perform significantly better than individual ones. Further example is the increased diagnostic performance of Mp-MRI in BC molecular subtype prediction based on the underlying biological features. For instance, well-known imaging biomarkers of triple-negative BC (TNBC) are intralesional necrosis and peritumoral edema at T2 weighted images, smooth margin and rim enhancement at CE-MRI Thus, the new perspective of breast MRI includes artificial intelligence (AI) applications. The intrinsic multiparametric nature of MRI has the greatest potential to incorporate AI applications into the so called precision medicine. The number of breast imaging biomarkers will increase in the next future, expanding the role of imaging in breast care.Medical imaging can be a source of biomarkers in diagnostic, predictive, prognostic and monitoring settings. assessing breast tissue vascular microenvironment and tissue permeability. As angiogenesis plays an important role in tumor biology, CE-MRI biomarkers and pharmacokinetic parameters were widely investigated. BPE is described as the enhancement of fibroglandular tissue in the dynamic CE-MRI early phases. In the last decade, BPE has generated interest and has been added in the last edition of BI-RADS MRI lexicon that provides standard descriptors for BPE level and distribution. It has been shown that BPE is a hormonally sensitive feature that declines over time with the onset of menopause, after oophorectomy and in patients who have been treated with tamoxifen or aromatase inhibitors. Initial results from two case–control studies on high-risk subjects have attested that BPE can be a predictive biomarker of BC risk. In particular, in females previously stated as at high risk of BC, a marked BPE increases the personal risk of BC up to tenfold. Kim et al found a significant correlation between qualitative BPE and epidermal growth factor receptor (EGFR)-positive BCs compared to EGFR-negative BCs. In this paper, BPE was also measured with the semi-quantitative background enhancement coefficient (BEC), evaluated through regions of interest drawn on healthy breast tissue. BEC as well as ipsilateral whole breast vascularity, were significantly higher in >2 cm tumors than in tumors smaller in size. In addition, BPE may reduce breast MRI sensitivity by obscuring enhancing cancers or may decrease specificity by determining enhancement patterns that mimic the appearance of malignancies. Nevertheless, current evidences have not confirmed a significant correlation between BPE and an increase in either false-positive or false-negative findings on breast MRI. However, a recent systematic review highlights the wide variability in the quantitative evaluation of BPE on breast MRI, thus uniform criteria should be defined to consolidate BPE as a biomarker.Over the past two decades, CE-MRI has improved breast MRI diagnostic accuracy with sensitivity up to 99% and variable specificities ranging from 47 to 97% in the detection and characterization of breast lesions, Breast lesions kinetic patterns differ between malignant and benign lesions; thus, enhancement time/intensity curve characteristics can be used in combination with morphologic features to improve differential diagnosis. Semi-quantitative parameters can be extracted from the enhancement curves, including the onset time, maximum signal intensity, gradient or rate of contrast uptake and washout, and initial area under the time signal curve. In recent years, the associations between contrast-enhancement kinetics and molecular subtypes were widely investigated. According to Blaschke and Abe, HER2 positive tumors demonstrated a faster and earlier enhancement than other subtypes, while luminal A and basal cancers showed a reduced washout during the delayed phase. This can be attributed to the frequent association of luminal A cancers with ductal carcinoma in situ, which rarely demonstrates washout kinetics; while basal subtype cancers are often characterized by tumoral necrosis and central scarring, which typically shows a persistent enhancement. Quantitative analysis involves pharmacokinetic modeling and requires more complex methods for estimating changes in tissue contrast agent concentration following intravenous injection. The transfer constant, Ktrans, describes the transendothelial transport of contrast medium by diffusion from the vascular space to the tumor interstitium and provides a measure of vascular permeability. Gradually, gadolinium diffuses back into the vascular system, with Kep representing the transfer constant from the interstitium to blood plasma and Ve the extravascular–extracellular volume fraction. Ktrans and Kep are generally high in tumors. A significant reduction up to a third has been detected in both parameters in patients with locally advanced BC early responding to NAT, while an increase in Ve has been shown in non-responders. According to the authors O’Flynn and Nandita M. de Souza, Ktrans can be used as a predictive biomarker to evaluate response to antiangiogenic drugs or vascular disruptive agents such as bevacizumab, a humanized monoclonal antibody directed against the vascular endothelial growth factor (VEGF), with a change in Ktrans value >40% commonly considered as the threshold for definitive disease response. In summary, the available literature shows an ample consensus on the diagnostic value of CE-MRI measurements for non-invasive characterization and prognostication of BCs as well as for therapy monitoring during NAT.Furthermore, in the era of new cellular signaling pathways and molecular therapies, CE-MRI can be used for quantitative assessment of the vascular microenvironment and the tissue permeability. DWI explores different functional tissue features including water molecules motion in the extracellular space, density of neoplastic cells, tissue microstructure, cell wall integrity and permeability. Compared to normal gland, tumor tissue is characterized by a lower water molecules diffusion and, consequently, lower ADC values due to the high cell density and the presence of numerous intra- and intercellular membranes, thus the ADC map allows to quantitatively evaluate diffusivity of water molecules. In recent years, the association between ADC value and standard histopathological and immunohistochemical breast tumor features such as histological type, grade, hormonal receptor and Ki-67 expression, HER2 status, were widely investigated. Bickel et al tested DWI as an imaging biomarker to differentiate ductal carcinoma in situ from invasive lesions. Authors demonstrated that ADC value −3 mm2/s allows the identification of invasive tumors with 78% sensitivity and 90% specificity  has established a multicenter, international working group composed of clinical experts, MRI physicists and MRI equipment suppliers with experience in breast DWI. DWI working group objectives include the promotion of DWI in MRI protocols, the diffusion of technical guidance for DWI protocols and the creation and improvement of quality control methods, to finally find agreement on the optimal image processing, visualization and interpretation. In a recent review, Iima et al addressed advanced DWI models, such as intravoxel incoherent motion (IVIM), diffusion tensor imaging (DTI) and diffusion weighted kurtosis (DKI). IVIM is a biexponential model that simultaneously evaluates tissue diffusivity and tissue microcapillary perfusion. DTI gives quantitative data on the water molecules directional diffusivity in biological tissues. The information obtained about diffusion anisotropy could be a potential biomarker of malignancy. It has been hypothesized that proliferating neoplastic cells, which generally destroy the normal structure of the mammary gland, could reduce anisotropy. DKI quantifies the incoherent movement of water molecules and tissue microperfusion typical of non-Gaussian phenomena, useful in the detection and characterization of breast lesions. A model proposed to quantify the Gaussian and non-Gaussian diffusion is able to estimate the D value, which represents the Gaussian diffusion, and the K value, a kurtosis parameter that represents the deviation from the Gaussian diffusion. These technological advances are supported by several studies and revealed to be useful in establish benign or malignant nature of breast lesions, in evaluating Ki-67 and tumor grading and in predicting treatment response. DWI is a promising qualitative and quantitative biomarker, a valid tool in lesion characterization and therapy monitoring. However, standardization of the acquisition and interpretation modalities of the extracted DWI data will enhance its clinical value.A review of the literature emphasizes DWI as a potential source of biomarkers to increase breast MRI specificity, significantly improving diagnostic accuracy and reducing unnecessary biopsies.cificity . In the in TNBCs .49–51 1H-MRS) based on the detection of the total choline peak (tCho) has been implemented in breast Mp-MRI protocols, since several studies reported higher levels of tCho in BCs compared to benign lesions and normal breast tissue. Therefore, on the basis of different choline peaks, MRS is a potential biomarker to improve diagnostic accuracy and predict tumor aggressiveness. The diagnostic accuracy of a high-spatial-resolution 3D 1H-MRS protocol at 3 T was evaluated for the characterization of breast lesions, based on tCho signal-to-noise ratio threshold levels and proved its potential in becoming a valuable adjunct to CE-MRI in distinguishing between benign and malignant breast lesions. Other authors demonstrated that tumor tCho measurements were significantly higher in invasive ductal carcinomas vs in situ cancers and that tCho correlated with numerous prognostic factors, including histologic and nuclear grade, and estrogen receptor status. Thus, the addiction of MRS in multiparametric protocol leads to significantly higher diagnostic accuracy than CE-MRI, also significantly reducing false-negative and false-positive cases. However, tCho sensitivity significantly decreases for smaller cancers, due to insufficient detection of tCho signal. Available data suggest tCho as a potential biomarker for treatment response assessment and early prediction of the final NAT effect. In treated lesions, an early decrease in tCho levels, after the initial course of therapy, is consistent with tumor response and is even more sensitive than other morphological and functional criteria. Instead, it was demonstrated an increase in tCho concentration in patients with local recurrence. Beyond tCho further metabolites can be detected and monitored, above all, the most promising seems to be the assessment of lipid metabolism. Thakur et al demonstrated that intratumoral lipid concentration allows to distinguish benign from malignant tumors and to differentiate among BC molecular subtype. Ramadan and colleagues described that healthy breast tissue in patients with BRCA-1 and BRCA-2 mutation was likely to differ from non-mutation carriers in unsaturated fatty acids triglycerides and cholesterol levels. Further studies are needed to support these results, that could open new scenarios in high-risk females screening programs. Recently, phosphorus spectroscopy (31P MRS) has been introduced as a new functional MRI parameter for BC diagnosis and therapy monitoring. In particular, it has been demonstrated that a decrease in the phosphoethanolamine/phosphocholine (PE/PC) ratio is a sensitive cellular-level indicator of malignancy. Moreover, changes in PE/PC ratios are observed during NAT. These promising results of 31P MRS have been obtained with 7 T-scanners. Thus, due to the lack of data collected with ultra-high field scanners certainly related to their limited diffusion, the use of 31P MRS as a breast MRI biomarker is still limited in clinical practice.MRS is a non-invasive functional technique that provides information on biochemical changes in specific anatomic structures by identifying and monitoring the chemical composition of the tissue under examination. In the last decades, proton MR spectroscopy  modalities, could predict the effectiveness of NAT and provide information on disease progression. Among AI applications specific methodologies are machine learning (ML) and DL. ML is a technology that allows the automatic training of machines with the aim of obtaining predictive data set based on the data and algorithms provided, without explicit programming. DL, a subset of ML, is characterized by a high accuracy, less need of human intervention but requires a huge amount of training data and expensive hardware and software. Due to MRI intrinsic multiparametric nature, ML application in breast MRI is fast-paced developing and many studies are demonstrating ML usefulness in lesion detection and classification, prediction of NAT response and recurrence risk, and therefore to guide therapeutic decisions. DL demonstrated high diagnostic accuracy to differentiate benign from malignant lesions, improving the diagnostic performance of breast MRI by decreasing the false positives and improving the positive-predictive value. Furthermore, DL has also been exploited extensively for evaluating the response to NAT.In a review published in 2018, radiomics models based on different imaging methods including MRI were investigated. Studies that analyzed BC using a radiomic approach and that provided data on BC diagnosis (detection or characterization), BC prognosis  or provided data on technical challenges  were included. Authors concluded that the application of radiomics in BC patients was an emerging translational research topic, with the capability of improving the knowledge of the breast lesions specifics. Nevertheless, AI applications are not ready to be incorporated into clinical practice, nor to replace expert human observers with the ability to critically evaluate MRI images and patient history.The integration of AI into breast imaging may enable the creation of new imaging biomarkers that incorporate patient clinical and tumor structural characteristics. Moreover, biomarkers could be incorporated into patient risk stratification via personalized imaging.Breast MRI may act as a diagnostic and prognostic tool to improve BC management through the extraction of a plenty of functional cancer parameters serving as imaging biomarkers. The intrinsic multiparametric nature of MRI provides specific information to visualize and quantify the functional processes of cancer development and progression, in order to improve detection and characterization of breast lesions, monitoring and prediction of response to therapy, and differentiation of biological BC subtypes. Moreover MRI images, due to their complex information content, are a fertile ground for AI applications. These may improve the integration of imaging biomarkers in clinical decision-making through the building of accessible predictive integrated models aiming at individualized medicine.


Setting up physiologically relevant in vitro models requires realizing a proper hierarchical cellular structure, wherein the main tissue features are recapitulated. Such a demanding goal necessitates providing cells with a biomimetic surrounding that enables their optimal adaptation in terms of overall cellular performance. Therefore, mimicking the extracellular matrix microenvironment, especially regarding physical and biochemical cues, is a prominent issue for optimal cellular spatiotemporal organization and differentiation. Many innovative approaches in cell culture are used to recapitulate a biomimetic surrounding, including bioreactors, microfluidic devices, biomaterial selection, biomaterial surface functionalization, micro- and nano-engineered scaffolds, responsive biomaterials, electrospinning, cells engineering, and 3D bioprinting. Besides promoting tissue morphogenesis and directing specialized behavior, these systems are reliable platforms for the observed cellular outcomes. For these main features, biomimetic-based systems are ideal for the drug testing/screening of new potential therapeutic molecules and/or for modeling a defined disease in a well-controlled environment. This Special Issue includes original research articles and reviews, providing critical and insightful views of the latest developments. The main aspects related to the realization of the 3D bioprinting of cell-laden scaffolds are provided in Zhang et al.’s review . The autZare et al.’s review  aims to Improved biomaterial biocompatibility is often achieved through different engineered surface strategies. Different surface functionalization approaches are described in a review by Sultana et al. , discussA new stent surface coating constituted by Mg/Al (LDH) nanoparticles loaded with anionic drug biochanin A and further encapsulated with heparin-tagged PLA–PEG copolymer was developed by Adepu et al. . The druLau et al.  comparedDash et al.  propose Ratlif et al.  presenteOverall, this Special Issue provides the latest insights in the regenerative medicine and tissue engineering fields; different approaches and methodologies have the common goal of creating a cell-friendly environment able to evoke optimal cell responses. The manuscripts in this volume also highlight the unmet clinical needs and the future challenges that must be faced for a more personalized path for restoring cellular and tissue functionalities. This knowledge is also helpful to emerging biomedical engineering areas such as smart and intelligent biomaterials, the biomanufacturing of cells and tissues, and personalized medicine.