Iron-rich food consumption has an invaluable effect for neonatal and fetal brain development as well as metabolic activities. Despite the public health importance of the consumption of iron-rich foods, there was no study, that assessed iron-rich food consumption in Rwanda. Therefore this study aimed to assess iron-rich food consumption and associated factors among children aged 6–23 months using Rwanda Demographic and Health Survey (RDHS).Secondary data analysis was done using RDHS-2019/20. Total weighted samples of 2455 children aged 6–23 months were included. Data coding, cleaning, and analysis were performed using Stata 16. Multilevel binary logistic regression were performed to identify factors associated with iron-rich food consumption. Adjusted Odds Ratio (AOR) with a 95% CI, and p-value <0.05 were used to declare statistical significance.The prevalence of good iron-rich food consumption was 23.56%. Northern province of Rwanda , mothers secondary education and above , married mothers , rich wealth status , having post-natal visit , mothers media exposure  and drugs given for intestinal parasite  were associated with iron-rich food consumption.This study shows that overall iron-rich foods consumption was low in Rwanda. The residing in the North province, mother’s secondary and higher educational status, married marital status, rich and middle wealth status, having media exposure, drugs given for intestinal parasites, and having child’s post-natal checkup were variables significantly associated with iron-rich food consumption. The region-based intervention will improve the consumption of iron-rich food. In addition, health policies and programs should target educating mothers/caregivers, encouraging parents to live together, improving their wealth status, working on mass media access by the women, and encouraging mothers post-natal checkups to improve iron-rich food consumption. Iron deficiency is the most common micronutrient deficiency, hitting more than two billion people worldwide, with African children bearing the most burden , 2. IronIron is an important element in brain metabolism –12. IronChildren under the age of seven are the population group most sensitive to iron deficiency, especially children under the age of two are vulnerable to iron deficiency because of their rapid growth , 14. As Low consumption of iron-containing foods and consumption of foods that interfere with iron absorption, such as phytates, also increase the risk of iron deficiency . Iron-riCurrently, IDA control methods include the use of iron treatment on a continuous or intermittent basis, the consumption of iron sprinkles or fortified foods and beverages, better food safety, and dietary diversity monitoring. There was no previous research on the consumption of iron-rich foods in Rwanda. Therefore, this study aimed to assess the prevalences of iron-rich food consumption and associated factors in Rwanda using the recent Rwanda Demographic and Health Survey (RDHS-2019/20), which has a paramount effect to develop intervention strategies based on the findings of the study to improve iron-rich food consumption across the country. This study may also assist policymakers, NGOs, global organizations, and researchers in identifying the factors in Rwanda that influence iron-rich food consumption to provide urgent interventional measures and resource allocation to enhance their behavior.In this study, the analysis was conducted based on large representative secondary data from Rwanda Demographic and Health Survey (RDHS) -2019/20, which was collected between November 9, 2019, and July 20, 2020. Rwanda is a landlocked country in Eastern Africa, bordered by Uganda, Burundi, Tanzania, and the Democratic Republic of the Congo, and is administratively subdivided into Kigali City and four provinces  . The 201The dependent variable of this study was the consumption of iron-rich foods by children aged 6–23 months, which was categorized as good and poor consumption. According to the DHS guideline, the number of youngest living children 6–23 months living with their mother who consumed at least one food rich in iron at any time in 24 hours preceding the interview among four food items, egg, organ meat, meat , and fish or shellfish were considered as good consumption, otherwise poor consumption .The individual-level variables are sex of the child, age of mother and child, level of education of mother and father, number of ANC visits, place of delivery, child postnatal check within 2 months, taking of drugs for intestinal parasites, and wealth status, whereas, the community-level variables include community-level poverty, media exposure, distance from the health facility, region, and residence which was driven from individual-level variables. After combining and recoding the respondents’ exposure to newspaper/magazine, radio, and television, community-level media exposure was developed. Because the data were not normally distributed, the median was used, and the results were classified as low if less than 50% of respondents were exposed to at least one medium, and high if more than 50% of respondents were exposed to at least one medium.In this study, Stata version 16 software was used for data analysis. Prior to analysis, the data were weighted to verify the DHS sample’s representativeness and to get trustworthy estimates and standard errors. Hence, the DHS data is hierarchical we used women’s individual sample weight (V005/1000000) throughout the analysis. For the descriptive results, we used cross-tabulations and summary statistics.DHS datasets contain hierarchical data structures with individuals nested under geographical clusters (primary sampling units) where children aged 6 to 23 months were nested within a cluster. This may violate the assumptions of standard logistic regression models such as the equal variance and independence assumptions. Thus, four models were fitted: the null model (no explanatory variables), model I , model II (community-level factors), and model III . Besides, the Intra-class Correlation Coefficient (ICC), Median Odds Ratio (MOR), and Likelihood Ratio test (LLR) values, as well as the deviation (-2LLR), were utilized for model comparison and fitness, respectively. Model III was chosen as the best-fitting model because of its lowest deviation.Random effects or measures of variation of the outcome variables were estimated by the median odds ratio (MOR), Intra Class Correlation Coefficient (ICC), and Proportional Change in Variance (PCV). Taking clusters as a random variable, the MOR is defined as the median value of the odds ratio between the area at the highest risk and the clusters at the lowest risk clusters when randomly picking out two clusters. variance , 32.The fixed effects or measure of association were used to estimate the association between the likelihood of prevalence of iron-rich food consumption and individual and community levels variables Finally, in the multivariable analysis, adjusted odds ratios with 95%confidence intervals and a p-value of less than 0.05 were utilized to identify associated factors of iron-rich food consumption.πij: the probability of iron-rich food consumption, 1−πij: the probability of iron-rich food consumption. ß0 is the intercept that is the effect on iron-rich food consumption when the effect of all predictor variables is absent. β1xij are individual and community level variables for the ith individual in group j, respectively. The ß’s are fixed coefficients indicating a unit increase in X can cause a ß unit increase in the likelihood of iron-rich food consumption. The uj shows the random effect (effect of clusters on the mother’s choice to give iron-rich food) for the jth clusters [Where, clusters , 32.www.measuredhs.com. All the ethical standards are available at https://goo.gl/ny8T6X.The ethical approval and permission to access the data were obtained from the DHS website In this study, a total weighted sample of 2455 children aged 6–23 months were included. The median age of children was 14 months (IQR = 10–19 months). The majority of the children(83.59%) were from rural areas, and the majority of them were from the east province(26.78). More than half of the children(50.55%) were males. The majority of the children (43.70%) were from poor households family .Due to the hierarchical nature of the DHS data, we assessed the clustering effect. The ICC in the null model was high in the random-effects analysis. This means that the variance between clusters accounted for around 19.89% of the variability in good iron-rich food consumption, whereas the remaining 80.11% was due to individual variation. The empty model’s higher MOR value suggested a significant difference in iron-rich food consumption between clusters. In the empty model, the MOR value was 2.36, showing that if we witnessed two children from two different clusters, a child in the cluster with high iron-rich food consumption was a 2.36 times higher likelihood of having good iron-rich food consumption as compared to a child within the cluster with lower iron-rich food consumption. Model fitness was also assessed using deviance, with the model with the lowest deviance chosen as the best-fit model, in this case, Model III with a deviance of. 2336.953 .The prevalence of iron-rich food consumption among children aged 6–23 months in Rwanda was 23.56% . Fish or shellfish was commonly consumed food (15.46%), whereas the liver, heart, and other organs were the least (1.22%) consumed iron-rich foods .From the final model, residing in the North province was the community-level variable positively associated with iron-rich food consumption. Similarly, mother’s educational status, marital status, wealth status, media exposure, drugs given for intestinal parasites, and child post-natal checkup were the individual-level variables positively associated with iron-rich food consumption.Accordingly, children who live in the North province were 74% less likely to consume iron-rich food  compared to those who live in Kigali city. Children whose mothers had secondary education and above had  higher odds of iron-rich food consumption compared to no education. Children whose mother is married had higher odds of iron-rich food consumption compared with unmarried ones ). Children from rich families had  higher odds of iron-rich food consumption compared with poor household families. Children whose mothers attended media had  higher odds of iron-rich food consumption compared to their counterparts. Children who received drugs for intestinal parasites had  higher odds of iron-rich food consumption compared to their counterparts. Children who had post-natal checkups had  higher odds of iron-rich food consumption compared to no post-natal checkups .Iron is a necessary micronutrient for a child’s growth and development. Due to an increased need for iron during periods of rapid growth, the World Health Organization recommends iron supplementation in young children . This stIn this study, residing in the North province was the community-level variable positively associated with iron-rich food consumption. Similarly, mother’s educational status, marital status, wealth status, media exposure, drugs given for intestinal parasites, and child post-natal checkup were the individual-level variables positively associated with iron-rich food consumption.This study revealed that the odds of iron-rich food consumption among children who live in the North province were lower compared to children who live in Kigali city. This might be due to the difference in lifestyle, sociocultural, and awareness of iron-rich foods, hence those who live in cities are more likely to have better access to media and other advanced websites, which ultimately increases their ability to read and comprehend the nutritional guidelines . This imThis study showed that the odds of iron-rich food consumption among children whose mothers were married were higher compared with unmarried mothers. Possible explanations could be that married mothers might have better coordination with their spouse, an increase in home effort, and receive better information from their spouse. This shows that encouraging parents to live together will improve the consumption of iron-rich food.This study revealed that the odds of iron-rich food consumption among children from rich families were higher compared with poor families. The possible explanation could be children from poor households have poor access to adequate food, which makes them unable to get diverse food sources for the consumption of iron-rich food . It indiThe odds of iron-rich food consumption among children with media-exposed families were higher compared to no media exposure. This is consistent with a study done in sub-Sahara Africa , and a sIn this study, the odds of iron-rich food consumption among children who had a post-natal checkup within 2 months were higher compared to their counterparts. This is in line with the previous study , 26, 45.The odds of iron-rich consumption among children who received drugs for intestinal parasites in the last 6 months were higher compared to those who did not receive drugs. This finding is in agreement with a study done in sab-Sahara Africa . This coRegarding the strengths, the study uses nationally representative data in Rwanda countries, which is representative across the countries. This study also used a multilevel modeling technique to provide a more credible result that takes into consideration the hierarchical nature of the survey data. However, the study is not free of drawbacks. The survey is susceptible to social desirability due to the self-reported nature of the interview, and the study’s cross-sectional design may not explain the temporal association of the independent and outcome variables. The other limitation of this study is it only accounts animal sources of iron-rich food, which may affect the prevalence of iron-rich food consumption.This study shows that overall iron-rich foods consumption was low in Rwanda. Residing in the North province, mother’s secondary and higher educational status, married marital status, rich and middle wealth status, having media exposure, drugs given for intestinal parasites, and having child’s post-natal checkup were variables significantly associated with iron-rich food consumption. The region-based intervention will improve the consumption of iron-rich food. In addition, health policies and programs should target educating mothers/caregivers, encouraging parents to live together, improving their wealth status, working on mass media access by the women, and encouraging mothers post-natal checkups to improve iron-rich food consumption.


Chronic graft-versus-host disease (cGVHD) is a serious complication after allogenic hematopoietic stem cell transplantation , negatively affecting the morbidity and mortality of recipients. Skin involvement is the most common cGVHD manifestation with a wide range of pleomorphic features, from scleroderma to ulcerations and microangiopathic changes. Despite the access to many immunosuppressive drugs, therapy for cGVHD is challenging. Systemic steroids are recommended as the first-line treatment; but, in steroid-resistant patients, extracorporeal photopheresis (ECP) remains one of the subsequent therapeutic options. Here, we present a case report of a 31-year patient suffering from advanced steroid-refractory skin and oral mucosa cGVHD who was spectacularly treated with ECP. It was the first time we observed such “overnight” resolution of the graft-versus-host disease syndrome. The present report proves the important role of ECP in the treatment of steroid-resistant cGVHD, especially when other immunosuppressive therapies have failed. Graft-versus-host disease (GVHD) is one of the leading causes of morbidity and mortality after allogeneic hematopoietic stem cell transplantation  . Its patDespite the wide access to many immunosuppressive drugs, the treatment of cGVHD remains a challenge. Steroids are still the first-line treatment; but, in steroid refractory patients, extracorporeal photopheresis (ECP) remains one of the subsequent therapeutic choices . It is aHere, we present the case of a 31-year-old patient suffering from steroid-refractory skin and oral mucosa cGVHD who promptly and spectacularly responded to ECP.A 31-year-old male patient underwent allo-HSCT from a 9/10 HLA-matched male unrelated donor for acute myeloid leukemia (AML) in June 2021. He was diagnosed with AML and translocation t six months earlier. The patient received induction chemotherapy consisted of daunorubicin and cytarabine. As a consequence, he achieved complete remission (CR) with negative measurable residual disease (MRD). The remission was consolidated by two cycles of high-doses of cytarabine. As a myeloablative conditioning, busulfan and cyclophosphamide were provided. For GVHD prophylaxis, he received cyclosporine with methotrexate as well as anti-thymocyte globulin (Thymoglobulin).The transplantation procedure as well as the early post-transplant period were unremarkable, and the patient was found to have full donor chimerism at discharge. CR was confirmed using repeated bone marrow examination, and cyclosporin was gradually reduced till complete withdrawal five months after allo-HSCT.On day +212 after transplantation, while being off immunosuppressants, the patient was urgently admitted for progressive weakness, watery diarrhea (up to 3000 mL of stool daily), severe abdominal pain, and nausea. Blood count was within the normal limits. In biochemistry, hyperbilirubinemia  with elevated levels of transaminases and alkaline phosphatase was detected. Clostridium difficile as well as other bacterial, viral, and fungal infections were excluded. Finally, he was diagnosed with late onset acute GVHD grade 4 according to the MAGIC classification . The patCouriel et al. [The efficacy of ECP in cGVHD is well-established and reaches up to 70% in skin manifestation and 63% in oral mucosa, but the efficacy differs between studies . Effectil et al. ; howeverl et al. .Jodele et al. [Of note is that the differential diagnosis of microangiopathic changes presented in our patient includes TA-TMA, although GVHD may also play a role in the pathogenesis of this complication . There ie et al.  incorpore et al. . Althouge et al. . Higher e et al. .One should remember that some drugs may have an impact on the development of microangiopathic changes. Calcineurin inhibitors, including cyclosporin A, tacrolimus, and sirolimus have been proven to increase the risk of TA-TMA, while steroids and MMF seem to be relatively safe in this aspect .The exact mechanism of ECP in cGVHD is not well-understood. Some data suggest that ECP-induced T-cell tolerance depends on T-cell apoptosis and induction of regulatory T-cells . The rolTo the best of our knowledge, an “overnight” improvement of cGVHD after ECP has never been reported before. ECP may remain a valuable therapeutic option, especially for patients with skin and oral manifestation. The treatment with ECP should be initiated as soon as possible in those with steroid refractoriness.


Solid oxide fuel cells (SOFC) are promising, environmentally friendly energy sources. Many works are devoted to the study of materials, individual aspects of SOFC operation, and the development of devices based on them. However, there is no work covering the entire spectrum of SOFC concepts and designs. In the present review, an attempt is made to collect and structure all types of SOFC that exist today. Structural features of each type of SOFC have been described, and their advantages and disadvantages have been identified. A comparison of the designs showed that among the well-studied dual-chamber SOFC with oxygen-ion conducting electrolyte, the anode-supported design is the most suitable for operation at temperatures below 800 °C. Other SOFC types that are promising for low-temperature operation are SOFC with proton-conducting electrolyte and electrolyte-free fuel cells. However, these recently developed technologies are still far from commercialization and require further research and development. The growing global energy demand coupled with the need to reduce emissions of environmentally harmful greenhouse gases have resulted in the search for new clean alternative energy sources. In this regard, fuel cells are attracting great attention. These are efficient and silent electrochemical devices that directly convert the chemical energy of a fuel into electrical energy without the limitations of the Carnot cycle. There are several types of fuel cells: alkaline fuel cells (AFC), phosphoric acid fuel cells (PAFC), molten carbonate fuel cells (MCFC), proton exchange membrane fuel cells (PEMFC), and solid oxide fuel cells (SOFC) . The higIntensive research of SOFC has been going on for three decades. During this time, many electrolyte and electrode materials have been studied, and a large number of SOFC configurations have been proposed and implemented. The first works ,4 in whiIn the presented work, we tried to collect all currently existing types of SOFC and highlight their advantages and disadvantages.The SOFC classification will be carried out according to several criteria: presence/absence of electrolyte, gas spaces separation, operating temperature, support types, and cell design. Individual fuel cells but not stacks will be used as the subject of classification, although it should be recognized that in many cases the advantages and disadvantages of a particular design are manifested precisely when cells are assembled into a stack.It is considered that the conventional SOFC structure is a three-layer one consisting of porous electrode layers (anode and cathode) separated by a dense electrolyte layer. However, in the last decade, electrolyte-free fuel cells (EFFC) have been developed. Sometimes, they are also called electrolyte-layer-free fuel cells, single-component fuel cells (SCFC), or non-electrolyte separator fuel cells (NEFC).Three-layer solid oxide fuel cells (TL-SOFC) are divided into two large classes according to the type of charge carrier in the electrolyte: oxygen ions or protons. SOFC with oxygen-ion-conducting electrolyte are the most developed and have reached the commercialization stage. Usually, the abbreviation SOFC means exactly this type of fuel cell, and it is used without any reservations, but sometimes, when compared with other variants, the oxygen-ion-conducting electrolyte SOFCs are designated O-SOFC . SOFC wi2− across the electrolyte, the oxygen on the cathode side must enter the electrolyte lattice from the gas phase, leave the electrolyte lattice on the anode side, and react with fuel. The cathodic reaction of converting O2 to O2−, known as oxygen reduction, involves the absorption of electrons, whereas electrons, H2O, and CO2 form at the anode when hydrogen or hydrocarbon fuel interacts with O2− supplied by the electrolyte. , and concη is the voltage due to concentration losses. The electrolyte layer makes the main contribution to ohmic losses, since its conductivity is 2–3 orders of magnitude lower than that of electrode materials . I. I50]. It 750 °C ,53. Onlyt 750 °C  was a spIn a mixed-reactant design proposed by M. Horiuchi et al. , the fueThe mixed-reactant fuel cells (SC-SOFC and DF-SOFC) have several advantages over DC-SOFC ,56, espeOn the other hand, mixed-reactant SOFC have serious drawbacks that impede their practical use ,56. ElecCurrently, DF-SOFC are fabricated based on oxygen-ion-conducting electrolytes . To makeAt the end of this section, it is worth mentioning the so-called flame-assisted fuel cells or flame fuel cells (FFC) ,66,67. TThe first SOFC operated at temperatures of 900–1000 °C . High opAt the present time, SOFC are usually divided into high-, medium-, and low-temperature categories. However, there is still no consensus on temperature ranges. In works ,70,71 SOThe temperature of 800 °C between high- and medium-temperature ranges, accepted by most authors, implies the upper limit of the expediency of using steel interconnects for the stack manufacture . In Ref.−2 . Sh. Sh75]. The concept of symmetric SOFC (SSOFC) is to replace different electrode materials (anodic and cathodic) of conventional SOFC on one material . This siAnother noteworthy concept is reversible SOFC (RSOFC or RSOC), which implies that a solid-state electrochemical device can operate both in the fuel cell mode and in the electrolysis mode ,160. In A brief description of all SOFC configurations developed today is presented in the review. To cover all SOFC concepts, the standard SOFC classification is supplemented by division according to such criteria as presence/absence of electrolyte and gas spaces separation. Herewith, the types of SOFC that are usually not mentioned in the classifications (electrolyte-free fuel cell and mixed-reactant SOFC) have been considered along with other types of SOFC from standpoint of standard criteria: operating temperature, support types, and geometry. This has made it possible to compare the various designs. It is shown that the most developed group of SOFC are separate-reactant fuel cells with oxygen-ion-conducting electrolytes. Among them, the most popular design is an anode-supported one, which permits one to achieve high specific powers at temperatures below 800 °C. However, electrolyte-free SOFC and proton-conducting electrolyte SOFC, the intensive development of which began recently, have a greater potential for reducing operating temperatures than standard dual-chamber O-SOFC. All SOFC types have some drawbacks; therefore, further research and new ideas are necessary for the practical mass implementation of this technology.


Multiple myeloma remains an incurable disease with the usual disease course requiring induction therapy, autologous stem cell transplantation for eligible patients, and long-term maintenance. Risk stratification tools and cytogenetic alterations help inform individualized therapeutic choices for patients in hopes of achieving long-term remissions with preserved quality of life. Unfortunately, relapses occur at different stages of the course of the disease owing to the biological heterogeneity of the disease. Addressing relapse can be complex and challenging as there are both therapy- and patient-related factors to consider. In this broad scoping review of available therapies in relapsed/refractory multiple myeloma (RRMM), we cover the pharmacologic mechanisms underlying active therapies such as immunomodulatory agents (IMiDs), proteasome inhibitors (PIs), monoclonal antibodies (mAbs), traditional chemotherapy, and Venetoclax. We then review the clinical data supporting the use of these therapies, organized based on drug resistance/refractoriness, and the role of autologous stem cell transplant (ASCT). Approaches to special situations during relapse such as renal impairment and extramedullary disease are also covered. Lastly, we look towards the future by briefly reviewing the clinical data supporting the use of chimeric antigen receptor (CAR-T) therapy, bispecific T cell engagers (BITE), and Cereblon E3 Ligase Modulators (CELMoDs). In the current era of multiple myeloma therapy, excellent treatment options exist for patients that result in survival of at least 5 years in about 55% of patients . HoweverRelapsed and/or refractory disease as defined by the International Myeloma Working Group (IMWG) is useful for designing clinical trials . “RefracIn this review of RRMM we discuss the pharmacologic mechanisms underlying the commonly used agents in the relapse setting which include immunomodulatory agents (IMiDS), proteasome inhibitors (PIs), monoclonal antibodies (mAbs), traditional chemotherapy, and the BCL2 inhibitor Venetoclax . We alsoThe introduction of immuno-modulatory drugs began with Thalidomide in the late 1990s for relapsed refractory multiple myeloma (RRMM) and revolutionized the treatment options offered to patients. A phase II study enrolling 84 RRMM patients led to the eventual approval of Thalidomide with Dexamethasone in 2006 by the FDA to treat both newly diagnosed multiple myeloma (MM) and RRMM . The earThe IMiDs share a common mechanism of action with newer generations showing added anti-myeloma activity and differing in pharmacologic properties such as half-life, liver metabolism, and the need for renal dosing ,7. As a +/CD8+ T cells which promotes immune surveillance and an anti-myeloma profile [+ T cells which may be an additional mechanism by which IMiDs may reverse such senescence and exhaustion [+ T cells, and effector memory CD8+ T cells were all increased following Lenalidomide treatment—suggesting immunomodulation on many different lymphoid compartments [In addition, immune dysregulation is a hallmark of MM by way of abnormal Th1/Th2 ratios, aberrant T cell function achieved via TGF-β secretion by MM cells, and immune suppression via disruption of Treg/Th17 balance . IMiDs i profile . Myelomahaustion . Lenalidhaustion . Myeloidartments .Phase 1 data regarding first-generation proteasome inhibitors (PIs) was first published in 2002 and paved the way for the eventual approval of Bortezomib in 2003 for RRMM . Data frThe pharmacology of PIs is related to their ability to inhibit NF-kB signaling through both canonical and non-canonical pathways via proteasome inhibition of IkB and p100/105 proteins, respectively . DownstrThe accelerated approval of Daratumumab, an anti-CD38 IgG1 monoclonal antibody, in 2015 for the treatment of MM marked the beginning of an era utilizing monoclonal antibodies to treat heavily treated MM patients . This apThere are several mechanisms by which Daratumumab functions as an anti-myeloma drug: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP) ,37,38,39+ NK cells directly towards SLAMF7+ MM cells and induces dose-dependent ADCC similar to Daratumumab without cytotoxicity to autologous SLAMF7+ NK cells [Hsi et al. found that SLAMF7 was expressed in a variety of immune cells including malignant hematopoietic cells, CD8 T cells, B cells, monocytes, dendritic cells, and most importantly, NK cells . Non-lymNK cells ,48. Not NK cells ,49,50,51NK cells .Traditional chemotherapy utilized in several malignancies is also used in MM in combination with therapies such as DT-PACE and CyBorD. The combination of Dexamethasone, Thalidomide, and the continuous infusion of Cisplatin, Doxorubicin, Cyclophosphamide, and Etoposide is coined DT-PACE. An additional and highly utilized regimen of Cyclophosphamide, Bortezomib, and Dexamethasone (CyBorD) is available for use and was recently trialed with Daratumumab with a durable response . The alkThe use of chemotherapy in combination therapy is based on synergy with each other. Cisplatin achieves cytotoxicity through the covalent binding of platinum to guanine and adenine resulting in intra and inter-strand crosslinking that promotes strand breaks within the DNA which results in cell apoptosis . HepaticHistorically having been approved for chronic lymphocytic leukemia and acute myeloid leukemia, Venetoclax has found a new use in those MM patients harboring a t(11:14) mutation . As an oSelinexor, is a first-in-class oral selective inhibitor of nuclear export (SINE), currently, FDA approved in combination with Bortezomib and Dexamethasone for patients having received one prior therapy . SelinexThe molecular mechanisms by which CAR-T and BITE therapies are effective in treating myeloma are largely based on the interaction of the malignant plasma cell with autologous T-cells. Multiple cell surface proteins expressed on plasma cells are targets for drug development with the most notable being B-cell maturation antigen (BCMA), a transmembrane, non-tyrosine kinase, glycoprotein. BCMA is an ideal target to inhibit as it is not only preferentially expressed on plasma cells with minimal expression in stem cells or non-hematopoietic tissue but is also needed for the survival of bone marrow plasma cells . FurtherBCMA CAR constructs contain an extra-cellular component derived from an immunoglobulin heavy and light chain variable domains that link to form a single chain variable fragment (scFv) capable of recognizing BCMA . A hinge+ and CD8+, leading not only to myeloma cell lysis but also differentiation of naïve T cells into memory T cells as well [BITE therapies are recombinant proteins that contain two separate linked single-chain variable fragments (scFv) which can simultaneously bind to a tumor cell and an immune effector cell to generate an immune synapse between the two . In the  as well .Lenalidomide is an established backbone of treatment for myeloma in both the front-line and maintenance settings. The development of resistance to lenalidomide is thought to occur due to the consequence of mutations in key signaling pathways in plasma cell clones that emerge after initial therapy . MultiplIn both doublet and triplet combinations, Pomalidomide has shown efficacy in the treatment of RRMM; however, clinical trials have varied in terms of the prior lines of therapy patients received prior to the introduction of POM. As a doublet, the Phase III MM-003 (NIMBUS) compared POM-loDEX vs. HiDEX and MM-010 (STRATUS) evaluated the effectiveness of POM-loDEX in a Phase 3b study ,76. In tp < 0.0001) as well in the LEN refractory patients  [As a triplet, the combination of POM-BOR-Dex was compared to BOR-dex in the phase III OPTIMISMM study in which the study population was enriched for patients considered LEN refractory, approximately 70% in the ITT population with 100% of patients having prior LEN exposure . At medi 0.0008) . Overall 0.0008) . In a mo 0.0008) .When considering the introduction of POM either as a doublet or triplet, the aforementioned clinical trials vary with respect to prior lines of therapy. In the STRATUS and NIMBUS trials, patients had received a median of five prior lines of therapy suggesting a role for POM in heavily pre-treated populations ,76. In cEmploying monoclonal antibody therapy with agents such as Daratumumab (DARA), Isatuximab (ISA) or Elotuzumab (ELO) either as a class switch strategy or in combination with POM are additional approaches that have been employed in the LEN refractory setting. There have been several clinical trials evaluating triplet combinations of a monoclonal agent plus proteasome inhibition and steroids. Where these trials differ is in the proportion of patients in each study considered LEN refractory. Given the activity of these monoclonal agents in the relapse setting, treatment decisions are often made based on pre-existing co-morbidities, prior drug exposure, patient tolerance, and insurance considerationsp = 0.0075) [p < 0.0001) [With respect to DARA, the CASTOR and CANDOR trials assessed the combination of DARA with either bortezomib or carfilzomib, respectively. In the Phase 3 CASTOR trial, patients were randomized to either DARA-BOR-dex versus BOR-dex with a crossover design in which patients in the control arm could receive DARA on progression . Patient 0.0075) . This be 0.0075) . In the  0.0075) ,81. This 0.0075) . In this 0.0075) . In the  0.0001) . Subgrou 0.0001) . Overallp = 0.0007) [With respect to ISA, the phase 3 IKEMA study compared the combination of ISA with Carfilzomib-dex (ISA-Kd) versus Kd as a control . In this 0.0007) . Similar 0.0007) . Similarp ≤ 0.3 the study did meet its primary endpoint, ; however, these results are signal generating and require larger randomized trials to validate [In terms of ELO, an initial phase 2 open label trial comparing ELO-BOR-dex to BOR-dex was completed with a primary endpoint of PFS. Based on a pre-specified significance level of validate . AdditioIn summary, the combination of Anti-CD38 monoclonal antibodies with proteasome inhibition and steroids shows efficacy in the RRMM setting in terms of PFS and ORR; however, an OS benefit is yet to be seen. Furthermore, it is important to note that subgroups of LEN refractory patients in these studies were relatively small and PFS benefit did not consistently reach statistical significance suggesting that these combinations may not be preferable for this group of patients. p = 0.0018) in APOLLO and 11.6 months (p = 0.001) in ICARIA, with control arms in both studies performing similarly with PFS of 6.9 and 6.5 months on POM-dex alone [The strongest evidence pointing towards clinical benefit in the use of monoclonal antibody therapy is in combination with POM-dex (Pd), specifically the Phase 3 APOLLO (Dara-Pd), Phase 3 ICARIA (Isa-Pd), and Phase 2 ELOQUENT-3 (Elo-Pd) studies. In all of these trials, patients had received a median of 2–3 lines of prior therapy and were compared to a control arm of POM-dex ,87,88. Iex alone ,89. In sex alone ,90.p = 0.028) at a median follow up of 35.3 months [In ICARIA specifically, the most recent survival data from the second planned interim analysis, published in March 2022, showed an OS difference of 6.9 months in the Isa-Pd versus the POM-dex control  [Interestingly, in ELOQUENT-3, at 45 months of follow-up, there was sustained, statistically significant separation of the curves in terms of OS for Elo-Pd versus Pd  . For the 0.0217) . StudiesPhase 3 clinical trials involving drugs with novel mechanisms of action  demonstrating benefit in LEN refractory RRMM remain areas of intense ongoing research. Further discussion regarding the role of cellular therapies targeting BCMA such as CAR-T and BITE therapies will be discussed in a later section of this review. The nuclear transport inhibitor, Selinexor (SEL), and the BCL-2 inhibitor Venetoclax (VEN), are two drugs with a role, particularly in the setting of early relapse.p = 0.0075). In the subgroup of patients with previous LEN exposure, the hazard ratio would suggest improved PFS but PFS duration or statistical significance was not reported [Regarding Selinexor, in the Phase 3 BOSTON trial patients were randomized to a combination of once-weekly SEL-BOR-dex (SVd) versus BOR-dex (Vd) with patients having received a median of one prior line of therapy . In thisreported . The finreported ,94. Whetreported . Additiop = 0.010) for the ITT population [p = 0.004) and OS , and ORR [Venetoclax, while not currently FDA-approved for RRMM, may have a role, specifically in patients with t or high BCL-2 expression. The phase 3 BELLINI study compared VEN-BOR-dex (Ven-Vd) versus Vd in patients having received a median of one prior line of therapy . Twenty pulation . Howeverpulation . In termpulation . When ev and ORR . Often when patients develop Bortezomib resistance/refractoriness, lenalidomide resistance/refractoriness is also encountered due to the nature of triplet and quadruplet therapies in the frontline setting incorporating both agents. The same strategies used to approach LEN resistance can also be used to approach BOR resistance; however, clinical trials that contain significant proportions of patients that are specifically BOR-resistant or dual PI-IMID resistant are lacking. These strategies involve the introduction of a second-generation proteasome inhibitor such as Carfilzomib (CAR) or Ixazomib (IXA), class-switching to a different class of drug with a novel mechanism, or the introduction of monoclonal antibody therapy. The presence or absence of specific cytogenetic abnormalities, previous therapy, and weighing risks/benefits in shared decision-making, guide treatment selection in this setting. p = 0.0045), median PFS , and ORR [The Phase 3 ASPIRE and ENDEAVOR trials established the role of Carfilzomib in the RRMM setting ,97. In A and ORR . Further and ORR . p < 0.0001) [p = 0.0017) [In the ENDEAVOR study, CAR was compared head-to-head against BOR, both arms in combination with Dex . Patient 0.0001) . Updated 0.0017) . These f 0.0017) .p = 0.01) [p = 0.495) [The oral proteasome inhibitor Ixazomib (IXA) was studied in the TOURMALINE MM1 trial in which the combination of IXA-LEN-dex (IRd) was compared to LEN-dex (Rd) with primary endpoint of PFS . Patient = 0.01) . Overall= 0.495) . Similar= 0.495) . Since T= 0.495) .Both the ASPIRE and ENDEAVOR trials inform decision-making as to whether Carfilzomib would be an appropriate choice in the RRMM setting, acknowledging its activity in patients having progressed on BOR, and side effect profile. It is clear from the ENDEAVOR data that switching to Carfilzomib is superior to re-treatment with Bortezomib. Ixazomib in combination with LEN-DEX provides an interesting, all-oral option for patients; however, survival data does not suggest sustained benefit long term. Additionally, to date, there have not been prospective randomized trials comparing Ixazomib to Carfilzomib or Bortezomib.Clinical trials previously mentioned in the context of LEN resistance, also apply to the BOR-resistant or dual PI-IMID resistant setting from the standpoint of incorporation of monoclonal antibody therapy or class-switching. Significant proportions of patients were considered BOR or PI refractory in the APOLLO, ICARIA, and ELOQUENT-3 studies. In APOLLO there was a trend towards improved PFS in both the BOR and dual refractory subgroups . SimilarLogically, given the efficacy of KRd, combinations of Carfilzomib-POM-dex or Carfilzomib-Cyclophosphamide-dex (KCyd) have also shown benefit in settings of BOR or dual BOR-LEN resistance, in Phase 2 trials. The final report from the Phase 2 EMN011/HOVON114 Trial, in which patients refractory to BOR and LEN received KPd, demonstrated an ORR of 92%, mPFS of 26 months, and mOS of 67 months . SimilarDaratumumab is currently approved for the treatment of multiple myeloma as both parts of initial induction and for relapsed disease. NCCN Consensus recommendations for induction regimens containing DARA for transplant-eligible patients include Dara-VTd, Dara-RVd, Dara-KRd, and DaraCyBorD. For transplant-ineligible patients, options include DaraRd, Dara-VMP, or DaraCyBorD. Given the more frequent use of DARA, resistance has inevitably emerged, and given the use of both IMID- and PI-containing regimens, it is becoming increasingly difficult to delineate which agent an individual patient with myeloma may be less responsive to. Furthermore, most Phase 3 clinical trials lack substantial subgroups of patients identified as DARA refractory making decision-making difficult. Reasonable strategies include re-treatment with DARA in cases of late relapse, or introduction of second-generation IMIDs or PIs depending on prior treatment exposure. Particularly in cases of early relapse after 1–2 prior lines of therapy, consideration of enrollment in clinical trials with novel cellular therapies such as CAR-T or BITE therapy should be considered. In situations where LEN or BOR are not implemented in first-line therapy, then logical choices include second- or third-line regimens that contain those agents in combination with DARA. Retreatment with DARA in combination with IMiDs specifically has been shown to overcome refractoriness to either agent in the frontline setting. For instance, Nooka et. al. performed a study in a series of 34 patients, the majority of whom were LEN and/or BOR refractory, separated into two cohorts-DARA and POM naïve or DARA-POM refractory . In bothThe antibody drug conjugate Belantamab Mafodotin (BEL) received accelerated FDA approval for RRMM based on results from the DREAMM-2 study published in 2020 . In DREAMore recently, Teclistamab (TEC), a bispecific T-cell engager, targeting BCMA on the plasma cell and CD3 on T cells, was studied in the Phase 1–2 MajesTEC-1 trial which led to its FDA approval for RRMM in patients experiencing progression after 3 prior lines of therapy including an Anti-CD38, IMID, and PI ,110. PatIf multiple lines of therapy have failed a patient, then multimodal chemotherapy is a reasonable option particularly if the disease is aggressive and immediate control is needed. These regimens often contain agents that a patient has not previously been exposed to in the front-line setting. While this strategy may not induce durable remissions, it will allow for disease control when patients require additional time for the production and manufacturing of a cell therapy product or as a bridge to autologous stem cell transplant. Regimens, or variations thereof, such as DT-PACE or DCEP have been shown to be effective in achieving rapid disease control ,112. In Choosing therapy in the Daratumumab refractory setting often implies refractoriness to multiple other agents including PI and IMiDs; clinical trial eligibility should be assessed for all patients in this setting. Decisions to re-treat with DARA, enroll in trials, pursue CAR-T, novel agents such as TEC, versus pursuing chemotherapy-based combinations are often decided based on the aggressiveness of the disease and the availability of a drug in a specific practice setting.In the relapsed myeloma setting there are two main situations in which there is a role for autologous stem cell transplant (ASCT): in patients who defer ASCT as consolidation in first remission or in patients who have relapsed after front-line ASCT during maintenance therapy. In p < 0.001) favoring frontline ASCT, but no significant difference in 5-year OS (p = 0.99) [p < 0.001) with median OS not reached in either group, and similar OS rates in the ASCT and no ASCT groups  [Highly effective therapies in the front-line setting, combined with patient preference, frailty, and comorbidities have raised the question in multiple studies of whether ASCT should be conducted in the front-line setting after induction or deferred to the time of first relapse. Data from the Phase 3 DETERMINATION trial showed at a median follow-up of 5 years, superior median PFS  . Similar = 0.81) . In IFM2009, achieving MRD negativity was a strong predictor of superior outcomes in terms of PFS, PFS2 (progression after next-line therapy), and OS . These MFor patients relapsed after the initial ASCT, the Myeloma X and ReLApsE trials represent the most mature data informing the second ASCT. In the Myeloma X trial, patients who relapsed after ASCT were reinduced with bortezomib, doxorubicin, and dexamethasone and subsequently randomized to ASCT with melphalan conditioning or weekly oral cyclophosphamide . While tRetrospective real-world data from the Center for International Blood and Marrow Transplant Research (CIBMTR) reported outcomes on 187 patients undergoing second ASCT for relapsed disease between 1995–2008 . PatientRenal impairment is a common feature of multiple myeloma, established as one of the “CRAB” criteria, present in about 50% of patients and associated with higher mortality . RegistrSeveral active agents in multiple myeloma can be safely dosed in patients with mild to moderate renal impairment. For patients with ESRD requiring dialysis, most medications can also be safely dosed post-dialysis to achieve a therapeutic effect. Out of all of the FDA-approved therapeutics mentioned in this review, generally, most IMIDs, PIs, and anti-CD38 monoclonal antibodies can be dosed safely in patients with renal impairment . ClinicaAlongside disease control and progression-free survival, achieving renal recovery with anti-myeloma therapy, measured as improvement in CrCl or eGFR by IMWG criteria, is of equal importance as this correlates with longer OS . The comWhile subgroup analyses assessing percent and time to renal recovery with DARA-based regimens have not been conducted from the pivotal Phase 3 CANDOR or CASTOR trials, there is real-world evidence and early phase trial data to support the use of Daratumumab in patients with renal impairment, including dialysis patients . In someLastly, a comprehensive evaluation for fitness for autologous stem cell transplant should also be undertaken in the treatment of RRMM. Renal impairment to any degree should not be a contraindication to ASCT as multiple studies have demonstrated that ASCT is safe and effective in patients with renal impairment, even on dialysis. A retrospective review of 475 patients from multiple bone and marrow transplant units in Vienna, Austria between 1998 and 2016 showed no difference in PFS for any stage of renal impairment and no difference in OS for eGFR as low as 45 . A similExtramedullary (EM) disease in myeloma occurs on a clinical spectrum that can range from bone-related plasmacytomas, hematogenous seeding of clonal plasma cells in soft tissue causing tumor formation in various organs, or plasma cell leukemia . ClinicaTreatment of patients who relapse with EM disease can be challenging as most clinical trials in myeloma exclude these patients, especially plasma cell leukemia. The same principles applied to the management of high-risk relapsed myeloma are applied to managing extramedullary relapse, including consideration of refractoriness to prior lines of therapy. In patients with diffuse visceral disease, rapid cytoreduction is needed and chemotherapy-based regimens such as VDT-PACE, BEAM, or HyperCVAD followed by ASCT or tandem ASCT-allo-SCT have been effective ,140,141.Relapse in the CNS, while uncommon, can occur in about 1% of RRMM patients, and the prognosis is extremely poor . ClinicaLocal radiation therapy or surgical resection should be considered in patients with symptomatic soft tissue or bone plasmacytomas at relapse. Radiation can be effective for local disease control and pain relief depending on the anatomic site. Additionally, surgical resection may be indicated if the plasmacytoma is causing mass effect, organ dysfunction, or axial skeleton or spinal instability. PET/CT imaging is instrumental in determining the extent of extramedullary relapse and monitoring response to therapy.The modern era of treatment in RRMM is rapidly evolving, with cellular- and immunotherapy being at the forefront of therapeutic innovation. Treatments such as chimeric antigen receptor T-cell (CAR-T) therapy, bispecific T-cell engagers (BITEs), and Cereblon E3 Ligase Modulators (CELMoDs) are demonstrating encouraging responses in the most heavily of pre-treated patients.−5 [−5 was seen in over 90% of patients [In terms of CAR-T cell therapy, there are currently two FDA-approved CAR-T products, both of which target anti-B cell maturation antigen (BCMA), idecabtagene vicleucel (ide-cel) and ciltacabtagene autoleucel (cilta-cel). For ide-cel, data from the Phase 2 KarMMa study, showed responses in patients having received a median of 5–9 prior lines of therapy, including an IMiD, PI, and anti-CD38 mAb . In the −5 . In term−5 . Cilta-c−5 . More re−5 . With lo−5 . MRD negpatients . On comppatients . Given tBITE therapy presents a versatile option for RRMM. Preclinical and early phase trials demonstrate efficacy as monotherapy which raises hypotheses of effectiveness in combination with traditional anti-myeloma therapy, as a bridge to transplant, or alternative to CAR-T cell therapy. Currently, there is significant energy towards the study of BITEs, with numerous Phase 1 and 2 trials including novel agents targeting plasma cell surface receptors BCMA, GPRC5D, FcRH5, and CD38, all paired with CD3 recognition to recruit T cells . Out of Lastly, CELMoDs, which are an evolving treatment strategy for myeloma, build upon the long-standing success of IMiDs by using a slightly different molecular structure that allows for more enhanced interaction with traditional IMiD substrates such as cereblon . Two ageDespite the promising success of these therapeutics, it is worth noting that in the real-world setting, outside of a clinical trial, access to these therapeutics can be difficult for patients. For instance, with CAR-T cell therapy, logistical issues related to time, CAR-T cell manufacturing, and institutional adaptation, limit its application in a timely manner if at all . FurtherHighly effective therapies for RRMM are helping to control the disease for our patients providing the benefits of improved survival and maintained the quality of life. Relapses continue to occur which is a humbling reminder that the disease remains much smarter than we are as clinicians and researchers. We have managed to outsmart this disease in some ways by leveraging our knowledge of the heterogeneity of plasma cell clones and markers of higher-risk disease and incorporating them into treatment decision-making.It is well established that next-generation IMIDs such as POM, next-generation PIs such as CAR and IXA, and monoclonals such as DARA, ISA, and ELO will continue to have substantial roles long term in the relapse/refractory setting. Other agents such as VEN and SELI are finding their own niche in very specific situations. Certain pillars of therapy such as ASCT and chemotherapy will continue to exist as options for the right patient in unique scenarios such as renal disease or extramedullary relapse. There is promise on the horizon as we race toward a functional cure for myeloma patients, with novel agents such as CAR-T and BITEs showing impressive activity in the most heavy of pre-treated patients, well beyond what was seen two decades ago. As myeloma is viewed more and more as a chronic disease, the key in assessing new therapies will be to answer questions not only related to patients’ survival and response, but also to their quality of life, reported outcomes, financial burdens, and disparities in access to care.


Current diagnostics of prostate cancer often show unsatisfactory results, leading to delayed detection or overtreatment. Urinary microRNAs are a class of promising non-invasive biomarkers. Although many studies have been conducted on this topic in the last five years, there is little agreement on the data obtained. This review aims to discuss new knowledge but also focuses on technical aspects affecting urinary miRNA analysis.Current routine screening methods for the diagnosis of prostate cancer (PCa) have significantly increased early detection of the disease but often show unsatisfactory analytical parameters. A class of promising markers represents urinary microRNAs (miRNAs). In the last five years, there has been an extensive increase in the number of studies on this topic. Thus, this review aims to update knowledge and point out technical aspects affecting urinary miRNA analysis. The review of relevant literature was carried out by searching the PubMed database for the keywords: microRNA, miRNA, urine, urinary, prostate cancer, and diagnosis. Papers discussed in this review were retrieved using PubMed, and the search strategy was as follows: (urine OR urinary) WITH (microRNA OR miRNA) AND prostate cancer. The search was limited to the last 5 years, January 2017 to December 2021. Based on the defined search strategy, 31 original publications corresponding to the research topic were identified, read and reviewed to present the latest findings and to assess possible translation of urinary miRNAs into clinical practice. Reviews or older publications were read and cited if they valuably extended the context and contributed to a better understanding. Urinary miRNAs are potentially valuable markers for the diagnosis of prostate cancer. Despite promising results, there is still a need for independent validation of exploratory data, which follows a strict widely accepted methodology taking into account the shortcomings and factors influencing the analysis. Prostate cancer (PCa) is estimated to be the second most often diagnosed cancer globally and the fifth most common cause of cancer-related death in men . It is tThe use of PSA as a biomarker for detecting prostate cancer caused a sharp increase in PCa incidence and lowered the number of metastatic or locally advanced tumors diagnosed . However, routine clinical screening also led to overdiagnosis and unnecessary treatment of indolent cancers that do not pose a threat to life or health. Furthermore, PSA is a prostate- but not cancer-specific marker, and it may be elevated in benign prostate diseases such as prostatitis or benign prostatic hyperplasia (BPH) . AdverseDespite advances in diagnosis implementing, for example, PSA derivatives, circulating tumor cells, cell-free DNA, circulating RNA, proteins, and peptides, there is a lack of biomarkers that would have sufficient sensitivity and specificity and would also be able to distinguish between different subtypes of PCa . CurrentmiRNAs are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. They are 20–24 nucleotides long and have an indispensable function in many biological processes such as cell development, differentiation and proliferation, DNA damage repair, apoptosis, and cellular communication . As one The first large-scale studies of dysregulated miRNA expression in prostate cancer tissue laid the foundations for a better understanding of miRNA involvement in prostate cancer pathophysiology. Such signatures can differentiate not only between tumor and non-tumor tissue but can also classify PCa according to disease aggressiveness. Most of these miRNAs regulate signaling pathways critical for prostate cancer, such as Wnt/β-catenin signaling  , but at the same time, it increases AR expression in PCa cell lines , potentiTumor-suppressor miRNAs, such as the miR-143/miR-145 cluster, often relate in prostate cancer to biological processes such as EMT, invasiveness, metastasis formation, or therapy response, which are typical of advanced stages of the disease. miR-143/miR-145 can suppress autophagy by downregulating ATG2B and thus sensitizing prostate cancer cells to radiation . MoreoveWith all that said, miRNAs are justly of scientific interest as a class of promising biomarkers in prostate cancer diagnosis and management. In addition to their intracellular roles, miRNAs are secreted by donor cells as a part of intercellular communication that includes regulation of gene expression in recipient cells. Cell-free miRNAs have been detected in different body fluids, including plasma, serum, and urine, and they show remarkable stability in both tissue and these so-called liquid biopsies. It has been proposed that miRNA can be released to the extracellular space by passive leakage from broken cells in cases of tissue injuries, chronic inflammation, etc., or they can be actively secreted by the cells in the form of extracellular vesicles (EVs), as well as bound to high-density lipoproteins.Compared to blood, the expression profiles of urine miRNAs in PCa have not been extensively studied, and most of the studies concerned focus on miRNA determination in extracellular vesicles, urine sediments, or whole urine . AlthougThe first study reporting changes in miRNA concentration in the urine of prostate cancer patients was published in 2012. Bryant et al. have quantified selected miRNAs, of which miR-107 and miR-574-3p were present at significantly higher concentrations in the urine of men with cancer compared to controls . These oUrine represents an easily accessible biological specimen and a source of extracellular biomarkers. Among the transcripts measured in urine as part of the combined panels currently approved for additional PCa diagnostics are the PCA3 and TMPRSS2-ERG tests; however, neither can significantly outperform the PSA test. The potential use of urinary miRNAs in clinical practice is supported not only by their biological characteristics but also by their exceptional stability and good analytical performance. When searching for new biomarkers, the most valuable are considered biomarker studies with large-scale screening (discovery phase) followed by one or more levels of validation. Such exploratory investigations provide a good basis for biomarker selection, but at the same time, they are financially and materially demanding. In their study, Byun et al. determined a miR-based urinary signature in urine supernatant from PCa patients using Agilent Human miRNA Microarray. On the subset of 12 miRNAs that showed significant differences between PCa and BPH samples, they used a ratio analysis, since urinary levels of upregulated miRNAs were compared with levels of downregulated miR-3659. For subsequent validation, the urinary miR-1913 to miR-3659 ratio was selected, where it was significantly higher in PCa than in BPH, with even improved diagnostic power within the PSA gray zone (AUC = 0.82) .Over a period of five years, several large-scale interconnected and innovative studies have been published. In the pioneer study by a team from Aarhus, Denmark, novel miRNA-based prostate cancer biomarkers were determined in cell-free urine. Using custom RT-qPCR array, urine miRNAs were profiled within a unique cohort counting more than 200 specimens. Instead of validating only significantly dysregulated miRNAs , the authors used the same technology also within a new independent cohort. They successfully validated six upregulated miRNAs and 22 downregulated miRNAs. In addition, they created diagnostic ratio models that compared all possible two-miRNA combinations. The diagnostic performance of all miRNA models was statistically evaluated and finally led to the construction of a definitive three-miRNA model (miR-222-3p*miR-24-3p/miR-30c-5p), which allows for distinguishing between PCa and BPH patients with an AUC of 0.95 but was also significantly associated with PC aggressiveness . To incrMeasurements in urine sediment allow for the detection of significantly higher levels of miRNAs; however, the content can change under pathological conditions. In 2021, Hasanoğlu performed a urine sediment and serum miRNA profile analysis uncovering a set of 49 differentially regulated miRNAs in samples from PCa patients compared to healthy controls. However, only miR-320a was validated as a valuable biomarker in early PCa diagnosis . In a piSome studies focus, due to the large amount of expression data already presented or stored within database repositories, on validating already published results. Investigating the most prevalent urological tumors, Ghorbanmehr et al. evaluated the urinary levels of miR-21-5p, miR-141-3p, and miR-205-5p in patients with bladder (BCa), prostate cancer, benign prostatic hyperplasia, and healthy controls. All selected oncogenic miRNAs appeared to be significantly upregulated in cancer-related samples with higher levels in PCa compared to BCa. Despite that these miRNAs were selected based on bladder cancer profiling studies, they showed good discriminating abilities within PCa diagnostics with the same sensitivity as PSA, but higher specificity . Based op = 0.034). Conversely, let-7c showed insignificant differences between PCa and HC in urine sediment, while in urine exosomes, this miRNA was not only upregulated, but also correlated with clinical stage (p = 0.023) Moreover, miR-21, miR-141, and miR-214 in urine sediment and miR-21, miR-375, and let-7c in urinary exosomes were associated with disease aggressiveness when significant deregulation in intermediate/high-risk PCa versus low-risk/healthy subjects was observed [It is the urine fraction used for miRNA analysis in which most of the published studies differ. For this reason, some publications include the concurrent analysis of several types of biological specimens ,36,42. FDespite a rather limited number of studies, there is a noticeable overlap between potential diagnostic miRNA. Among studies conducting a large-scale screening, miR-30b, miR-31, miR-24, and miR-125b were described as dysregulated in two studies ,35 , cell-depleted urine samples (urine supernatant) are used in the following studies. The isolation of miRNAs is usually performed using commercial isolation kits. Most analyses are based on RT-qPCR experiments that quantify selected miRNA targets in the exploratory cohort. Fewer studies employ high-throughput technologies, such as NGS or microarray profiling, to identify a higher number of miRNAs. In some studies, detected miRNAs are verified during the validation phase, which involves a larger set of patients see .Using hydrostatic filtration dialysis to isolate EVs, Xu et al. focused on qPCR analysis of the selected miRNAs. Of the four miRNAs measured in the exosomal fraction of the urine supernatant, miR-145-5p and miR-1290 were significantly increased in the group of 60 PCa patients in comparison to the group of 37 BPH patients. The study also demonstrated an increased diagnostic potential of miR-145-5p in combination with the PSA value (AUC = 0.863). Quantification of the other two targets, miR-572 and miR-141-5p, revealed significant differences in exosomal concentration between PCa patients and healthy controls, but not between the BPH and PCa groups . A new aAnother study explored dysregulated miRNA levels in a moderately large cohort of 80 urine samples. In this study, Danarto et al. showed significant differences in miR-21-5p levels between the groups of metastatic and non-metastatic PCa and BPH patients. Statistically significant was also the downregulation of miR-200c-3p in both PCa groups versus the BPH cohort . EmployiWani et al. focused on the analysis of exosomal miR-2909 and miR-615-3p in a set of patients with PCa, BPH, and bladder cancer patients. While their findings showed a markedly increased amount of miR-615-3p in the exosomes derived from both bladder or prostate cancer urinary samples compared to healthy or BPH patient specimens, miR-2909 was found exclusively in the exosomes of prostate cancer patients. Although in vitro experiments showed its high expression in bladder cancer cells, miR-2909 was absent in the exosomes derived from these cells. In addition to its specificity to PCa, miR-2909 showed a significant correlation with clinicopathological parameters, and it could differentiate patients into three PCa risk groups .p < 0.05). Furthermore, two candidate miRNAs, miR-30b-3p and miR-126-3p, confirmed their discrimination power in the independent cohort by outperforming serum PSA [Matsuzaki et al. performed a miRNA analysis in EVs extracted by differential centrifugation from whole urine obtained after DRE. miRNAs were detected using a hybridization microarray in a cohort counting 10 PCa patients and four subjects with negative biopsy. Overall, 19 miRNAs showed a significantly higher level in PCa patients  were validated by RT-qPCR reactions in the training cohort. The four-miRNA-based classifier was performed with 91% sensitivity and 89% specificity (AUC = 0.979) to distinguish PCa patients and healthy donors. Moreover, miR-375 separated localized vs. metastatic PCa samples, and in combination with miR-451a, it could differentiate PCa from BPH patients .A multi-marker approach is used within the Sentinel platform, which consists of three different tests that are based on the exosomal short non-coding RNAs (sncRNAs) isolated from the urine supernatant. The platform is based on a classification algorithm that reflects the sncRNA expression signature of the tested individual. The discovery phase (PCa and CS test) involved 235 participants and was performed using Affymetrix miR 4.0 arrays. Training and validation included a case-control sample of 1436 subjects, and the validation was performed using the OpenArray platform. Sentinel PCa test based on 85 miRNA and 60 small nucleolar RNA (snoRNA) targets could distinguish between PCa patients and PCa-free individuals with 94% sensitivity and 92% specificity. The Sentinel HG test (122 miRNAs and 25 snoRNAs) is aimed at men diagnosed with PCa to differentiate between lower (GG1-2) and higher-grade disease (GG3-5) with 94% sensitivity and 96% specificity. The Sentinel CS test (130 miRNAs and 66 snoRNAs) can be used to monitor the patient over longer periods, as it can classify low-grade (GG1) and intermediate- and high-grade cancer (GG2-5) with 93% sensitivity and 90% specificity .Due to the distinct methodologies used for EVs isolation and miRNA detection ,54,55,56As suggested before, miRNA analysis in urine suffers from shortcomings that are mainly connected to procedures within the preanalytical phase, e.g., urine collection, sampling, preservation, or storage. Here, the solution should be not only to optimize the preanalytical process, but also to recognize all biological and technological factors that affect miRNA analysis in urine and include them in data evaluation. The second important cause of inconsistency among studies is the detection itself, as every analytical method has its own limitations. In order to eliminate these limitations, new alternative detection methods or approaches could be implemented.One of the important factors not studied often within urine miRNA analysis is the effect of anti-cancer treatment including surgical procedures. Konoshenko et al. evaluated the effect of radical prostatectomy on the level of diagnostic miRNAs identified in their previous research . All 19 Another major factor that needs to be addressed is the intraindividual variability of miRNA levels in urine. While interindividual variability displays how differently a clinical biomarker is expressed between compared groups, intraindividual variability shows changes in the levels within one subject across repeated measurements. Therefore, a reliable biomarker should show low intraindividual variability and stability over time. This parameter is studied within the scope of protein biomarkers , but itsSince hematuria frequently occurs during prostate cancer , hemolysUrinary EVs represent a remarkable source of potential biomarkers since they reflect the molecular nature of the parental cells . MoreoveThe isolation methods for EVs vary among the studies, but precipitation-based techniques are used most often. The choice of separation technique may influence the characteristics of EVs, their analysis, and the presence of co-isolates such as uromodulin ,73. PolyAt present, the most common techniques for miRNA quantification are qPCR-based methods. Although these method are the gold standard, they have several disadvantages that can make miRNA detection quite difficult or inaccurate. In general, inconsistencies across the literature are not observed within the technical design, but rather within data processing and analysis, because no consensus on the process has been adopted. To improve this, a coherent and sequential procedure for data evaluation could be useful. From this point of view, Bryzgunova et al. presented a new perspective on PCa classification using urine miRNAs. Based on their extensive research in this field, they proposed a four-block data analysis algorithm that can be described as a series of steps that allow for the classification of suspected prostate cancer patients. The expression data of selected urine miRNAs and their ratios are analyzed according to pre-defined arbitrary cut-off ranges, which should compensate for the heterogeneity of the miRNA expression but also of the group population. Subjects are classified into these four steps as “Prostate disease”, “PCa”, “Not PCa”, or “Healthy” with a total accuracy of 97.5% . Often mIn order to achieve the detection of miRNA in urine at a lower concentration or to simplify the analysis, several alternative approaches have been proposed over the last five years. The main advantage of these methods is the direct detection of RNA without the need for reverse transcription and further amplification. In their study, Lee et al. focused on miRNAs in urine exosomes, which represent a promising source of cancer-related markers but are, however, isolated in laborious and multi-step procedures. To simplify the process, they demonstrated in situ detection using bi-labeled hairpins (molecular beacons) that include complementary sequences to miR-375 and miR-574-3p, which are both proposed markers of prostate cancer. Given their nano-size, the molecular beacons are able to penetrate exosomes where they hybridize with the miRNA strand and produce a fluorescent signal. Although this detection system holds considerable potential, the question of how urine affects individual parts of molecular beacons needs to be answered . To overUsing electrochemistry, Kim et al. performed the detection of miRNAs in urine from PCa patients with a graphene-based electrical sensor connected to a field-effect transistor. Peptide nucleic acids within this sensor chip hybridize with miRNAs in a biological sample and mediate a change in electrical signal. The tested molecules miR-21, miR-1246, and let-7b showed a significantly higher level in patients with prostate cancer compared to patients without the disease. Due to the label-free detection, good durability, and the dynamic range, this device could serve for rapid diagnosis based on urine miRNA assessment .p < 0.05) in the urine samples from PCa patients when compared to healthy controls. Moreover, nanostructures allowed for miRNA quantification without any pre-processing step, as it would be required for instance in PCR-based methods [Another new approach to analyze EV-associated miRNAs in urine is to use nanotechnology. One of the proposed systems employs an electrostatic collection of EVs based on the interaction of the negatively charged surface of urine EVs (pH 6–8) and positively charged nanowires. After in situ extraction, EV-encapsulated miRNAs can be analyzed using standard high-capacity screening methods, as in the case of Yasui et al., who performed a hybridization miRNA microarray and unveiled a large number of miRNAs dysregulated in PCa samples in comparison with non-cancer samples . Nucleic methods .Sometimes, the pathophysiology of the tumor is so complex and heterogenous that one class of biomarkers does not sufficiently display various aspects of the systemic response to a present tumor. For this reason, efforts have been made to develop multi-marker systems. Davey et al. proposed a panel that combined several mRNA and miRNA targets isolated from urinary EVs. To simplify the analysis, they utilized Vn96 peptide-mediated EV capture technology followed by qPCR-based detection of selected markers. The panel consisting of FOLH1, HPN, CD24, TMPRSS2-ERG, ITSN1, ANXA3, SLC45A3, miR-375, and miR-574 distinguished prostate cancer from benign states with an AUC of 0.843. The combination with PCA3 and clinical–pathological data improved the accuracy of the multi-marker system to an AUC of 0.955 . New altDespite a large amount of experimental data, published results need to be independently validated so that urinary microRNAs can be implemented as standard clinical biomarkers for prostate cancer diagnostics. The main reason is inconsistencies across studies, whether they differ in sample manipulation, the technical aspect of analysis, or subsequent data evaluation. One way to refine the analysis of miRNAs in urine is to focus on the molecules encapsulated within extracellular vesicles, because this fraction represents a discrete department of which the content is not easily affected by the urine environment. However, the analysis of EVs has several shortcomings mainly related to vesicle isolation, which, depending on the methodology used, achieves various efficiencies. For each urine fraction, qPCR remains the standard method for miRNA analysis, even if additional steps  are necessary and data evaluation is dependent on correct normalization. The solution could be provided by new methods based on different physical–chemical principles , which enable quick miRNA assessment in biofluid without previous manipulation with the sample. Despite their considerable potential, these methods need to be properly validated. Another approach is represented by multi-marker systems that combine biomarkers with different biological behavior. However, no method provides accurate results if it is performed on biological material of poor quality. Therefore, special attention should be paid to the pre-analytical phase, which plays a key role in the reproducibility of the results. Only a strict adherence to the widely accepted principles of miRNA analysis guiding pre-analytical steps, but also study design, and the analysis itself, can ensure production of the reliable data.


There was no difference between the groups in the incidence of ischemic stroke  and intracranial hemorrhage . Permanent pacemaker insertion was observed more frequently in the TAVR cohort , whereas repeat procedures were rare in both treatments . In the nation-wide real-world data analysis, TAVR with current-generation devices showed significantly lower 1-year mortality compared to SAVR in severe AS patients.Few studies have reported comparisons of out-of-hospital clinical outcomes after transcatheter aortic valve replacement (TAVR) and surgical aortic valve replacement (SAVR) in patients with severe aortic stenosis (AS) in the era of current-generation valves that reflect the real-world situation. Data on patients with severe AS aged 65 years or older who underwent TAVR or SAVR between 2015 and 2018 were obtained from the National Health Insurance Service in Korea and clinical event rate was analyzed. The primary endpoint was all-cause death at 1 year. The cohort included a total of 4623 patients over 65 years of age, of whom 1269 (27.4%) were treated with TAVR. After 1:1 propensity score matching, 2120 patients were included in the study. TAVR was associated with reduced 1-year mortality (hazard ratio (HR): 0.55; 95% confidence interval (CI): 0.42–0.70;  Transcatheter aortic valve replacement (TAVR) is now accepted as the preferred treatment over surgical aortic valve replacement (SAVR) for very old patients with symptomatic severe aortic stenosis (AS) ,2. HowevAlthough TAVR was introduced into global clinical practice more than 20 years ago, it was not introduced in Korea until 2012. Moreover, the number of TAVR cases only began to exceed 100 per year in June 2015, when national insurance began to pay partial costs of TAVR . At presLarge-scale randomized controlled trials (RCTs) generate robust statistical evidence and play a pivotal role in evidence-based medicine. However, most large-scale RCTs that compare TAVR- and SAVR-excluded populations with serious illness at the time of screening. Excluding patients with severe comorbidities from RCTs limit the generalizability to real-world clinical practice, especially for fragile geriatric patients who can benefit from less invasive treatment modality. In this regard, analyzing clinical results in real-world clinical practice is important for establishing a strong basis for treatment policies. Furthermore, there is a lack of large-scale clinical data for the relative performance of TAVR versus SAVR in Asian populations. Accordingly, the aim of this study was to compare the clinical outcomes of TAVR and SAVR performed in real-world severe AS patients using the current-generation devices in a broad range of ages.The primary endpoint was all-cause death. Ischemic stroke or intracranial hemorrhage (ICH) were analyzed as secondary endpoints. Permanent pacemaker insertion (PPI) and repeat AVR were assessed as safety endpoints.Data were collected from January 2015 to December 2018 from the National Health Insurance Service (NHIS) program (NHIS-2020-1-177). The NHIS is a mandatory health insurance program provided by the Korean government, covering almost 98% of the Korean population . The NHIThis is a retrospective cohort study and included patients who received a TAVR or SAVR between January 2015 and December 2018 identified from the Korean NHIS database, and the date of the earliest AVR during the period was set as the index date. To restrict the cohort to patients who received AVR for native valve, those with codes of repeated AVR were excluded from the analysis cohort, and to make study even more comparable, only bioprosthetic valve surgery was included. Additionally, patients who had other indicators of AVR, such as aortic regurgitation (AR), infective endocarditis, and cardiovascular infection , or underwent surgery with mechanical valve during index hospitalization were excluded. On the contrary, because TAVR is only performed in severe AS patients meeting stringent reimbursement criteria compiled in Korea, no patient who underwent TAVR was excluded on the basis of diagnosis code. Finally, patients younger than 65 years were excluded .Baseline characteristics were measured to improve the comparability of both treatment groups. Demographic characteristics included age, sex, and clinical characteristics, including comorbidities, such as hypertension, diabetes, heart failure, coronary artery disease (CAD), previous percutaneous coronary intervention (PCI), dyslipidemia, previous stroke, aortic disease, peripheral artery disease (PAD), atrial fibrillation (AF), chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD), and end-stage renal disease (ESRD).t-test. However, if continuous variable was not normally distributed, it was shown as median and IQR (interquartile range), as well as being compared using Mann–Whitney test. We performed a 1:1 propensity score matching (PSM) for the patients of both groups using a caliper width of 0.25 to reduce selection bias and expressed standardized mean difference. The propensity score was calculated using baseline characteristics, including sex, age, and underlying comorbidities . In the matched cohort, the time to clinical event at 1 month and 1 year were assessed using a Cox proportional hazards model, and the estimates were presented as hazard ratios along with 95% confidence intervals. The log-rank test was performed to assess if there was a significant difference in Kaplan–Meier survival curves between the TAVR and SAVR, and the Schoenfeld test was performed to assess if the hazards were proportional over time. In all comparisons, p-values < 0.05 indicated statistical significance. These statistical analyses were performed using the SAS Enterprise Guide version 7.1 .Descriptive statistics were calculated to show the differences in baseline characteristics of patients in the TAVR and SAVR cohorts. Categorical variables, expressed in percentages or frequencies, were analyzed using chi-square test or Fisher’s exact test as appropriate. Continuous variables, such as age, were expressed as mean ± standard deviation and compared using the unpaired Student’s Between 2015 and 2018, a total of 10,166 patients who underwent SAVR or TAVR were identified by previously depicted code extracting  from thep < 0.001; 80, 7 vs. 74, 8, p < 0.001). The female patients showed slightly higher trend in the TAVR group . The proportion of comorbid diseases, except for AF, was also higher in the TAVR group. Baseline demographics and clinical characteristics of selected patients are summarized in Patients in the TAVR group were significantly older than patients in the SAVR group , and the median ages were 79, 6 (IQR) in both groups (p = 0.203). The proportion of female patients was 52.5% in both groups. The proportion of any comorbidities did not differ statistically. The prescriptions of antithrombotics were analyzed and shown in A total of 1060 patients were eligible for 1:1 PSM from the TAVR and SAVR groups, respectively, resulting in balancing the distribution of baseline characteristics between the groups . The meaConcomitant coronary revascularization was performed in 12.9% of the patients in the TAVR group and 17.5% of the patients in the SAVR group. Other procedures in the surgery group included valve surgery involving valves other than aortic valve (AV), aortic graft replacement, myectomy, and atrial septal defect closure. In the TAVR group, the transfemoral approach was used in 97.8% of the patients. Balloon-expandable valves were used in 59.7% of the patients with valve systems identified in the TAVR group, and self-expandable valves were used in 37.8%. Details regarding the procedures are provided in p < 0.001) (p = 0.743) and intracranial hemorrhage  were not different between the groups. PPI was performed more frequently in the TAVR group  compared to the surgery group, while the risk of repeat procedure was rare and did not differ between the two groups . The 30-day outcomes are consistent with 1-year outcomes. The 30-day all-cause mortality was lower in the TAVR group , and permanent pacemaker implantation occurred frequently in patients who underwent TAVR . The Kaplan–Meier estimates of each clinical event are shown in TAVR showed lower all-cause mortality compared with SAVR at 1 year : 0.42–0.70, < 0.001) . The risThe major finding of this study is that 1-year all-cause death was significantly lower in patients aged 65 years or older undergoing TAVR for severe AS than with SAVR. TAVR showed significant differences in 30-day mortality at the beginning of the procedure and maintained this trend for up to one year. Among large-scale RCTs, only the CoreValve High Risk trial demonstrated that TAVR significantly reduced the all-cause death compared with SAVR at one-year outcomes, where old-generation self-expandable valves were used [Several large registries have not yet reported the benefit of the TAVR when extending to out-of-hospital follow-up. The reports of most registries to date show that mid-term performance of TAVR is comparable or inferior to surgery. Data from the GARY , the world’s largest TAVR registry, indicate one-year results for TAVR and SAVR were similar, while five-year results by PSM showed an inferior outcome of TAVR . The OBSOur findings may be the first to demonstrate superiority of TAVR over SAVR in reducing mortality at one-year follow-up in a nation-wide observational data. The superiority of TAVR in this study is believed to be due to several factors. First, this study enrolled patients who underwent TAVR using mostly new-generation valves, while previous studies recruited patients treated with old generation valves. If procedural skill improvement, the so called “learning curve effect”, dedicated patient selection, and periprocedural management have a great influence on in-hospital outcomes, more sophisticated contemporary valve systems are also thought to contribute to out-of-hospital outcome improvement ,21,22. IIn this study, the surgery group showed numerically higher mortality than that observed in previous studies ,26. We aAlthough large-scale RCTs for patients at various surgical risk levels demonstrated acceptable efficacy and safety outcomes with TAVR, TAVR has not yet been established as a standard treatment for relatively younger patients. This is due to the lack of long-term evidence of efficacy and safety of TAVR compared to SAVR ,28. EvenThis study provides new information about TAVR. First, the initial survival gain of the transcatheter procedure has sustained to out-of-hospital follow-up until one year by Kaplan–Meier analysis, suggesting a rationale for prioritizing TAVR in the treatment of severe AS. Second, TAVR can be a more beneficial treatment in Asian people. Third, there were no significant interactions between treatment modality and underlying co-morbidities that had been excluded from major RCTs, such as dialysis. Fourth, as is well known, TAVR was associated with a higher incidence of PPI. However, the absolute incidence of PPI in this study was relatively low compared to previous reports, even more so considering that the TAVR group contained about a third of patients with self-expandable valves ,30. It ip = 0.004). On the contrary, repeat AVR was excluded in SAVR; however, this was not the case in TAVR, which was disadvantageous to TAVR. Additionally, since concomitant procedures were collected during the index hospitalization period, it should be considered that there are cases that were not performed simultaneously on the same date. Third, valve size profile, key biochemical and imaging data for paravalvular leakage, PPM, and structural valve degeneration or bicuspid etiology were unavailable [This study has several limitations. First, the dataset analyzed was from the health insurance system in which researchers use codes to include or exclude patients and analyze clinical events. However, incorrect entry of disease codes is a serious shortcoming of the insurance data-based study. To minimize these shortcomings, we used claims codes and death events that are unlikely to differ from the actual practice and situation. Claims codes are heavily scrutinized and the information on survival or death is accurately monitored by the HIRA in Korea because it is related to financial reimbursement. Whereas many studies based on insurance data reported to date are dependent on disease codes, this study has a unique advantage in that it used claims codes. Second, ideal comparison of both treatment can be performed under the setting of isolated AVR. However, concomitant surgery, such as coronary artery bypass graft, valve surgery not for AV made the SAVR cohort unfavorable compared to TAVR, where PCI was a mainstay of concurrent procedures. Moreover, the fact that the frequency of coronary revascularization was higher in the surgical group also adversely affected the SAVR (vailable ,35,36,37vailable . Longer vailable ,39,40. FIn spite of the aforementioned limitations, current-generation device using TAVR significantly reduced all-cause death compared with SAVR in severe AS patients over 65 years old at 1-year follow-up from this nation-wide real-world analysis. A higher rate of permanent pacemaker insertion is associated with TAVR, but the absolute rate was reasonable.In real-world retrospective data, TAVR showed similar or inferior mid-term outcomes compared with SAVR in various nation-wide studies. However, retrospective studies comparing TAVR with SAVR in patients with severe AS were mostly performed with old-generation valve systems. In particular, large-scale data of Asian people for comparison of TAVR versus SAVR were scarce. In this Korean nation-wide real-world data analysis, TAVR with current-generation devices showed lower mortality after mid-term follow-up compared to surgery in severe AS patients, despite the higher PPI rates.


The accumulation of sequencing data has enabled researchers to predict the interactions between RNA sequences and RNA-binding proteins (RBPs) using novel machine learning techniques. However, existing models are often difficult to interpret and require additional information to sequences. Bidirectional encoder representations from transformer (BERT) is a language-based deep learning model that is highly interpretable. Therefore, a model based on BERT architecture can potentially overcome such limitations.Here, we propose BERT-RBP as a model to predict RNA–RBP interactions by adapting the BERT architecture pretrained on a human reference genome. Our model outperformed state-of-the-art prediction models using the eCLIP-seq data of 154 RBPs. The detailed analysis further revealed that BERT-RBP could recognize both the transcript region type and RNA secondary structure only based on sequence information. Overall, the results provide insights into the fine-tuning mechanism of BERT in biological contexts and provide evidence of the applicability of the model to other RNA-related problems.https://github.com/kkyamada/bert-rbp. The datasets underlying this article were derived from sources in the public domain: .Python source codes are freely available at Bioinformatics Advances online. Interactions between RNA sequences and RNA-binding proteins (RBPs) have a wide variety of roles in regulating cellular functions, including mRNA modification, splicing, translation and localization  is widely used to reveal a comprehensive picture of RNA–RBP interactions , have achieved state-of-the-art performance in various tasks  and SEP (separation). In our model, CLS was appended at the beginning of each input sequence, and its feature vector from the final layer was used for classification. The SEP was attached only to the end of each sequence.Attention represents the flow of information within the BERT model. The attention weight indicates how much information the hidden state of a token in the upper (closer to the output) layer referred to the hidden state of a token in the lower (closer to the input) layer.E-value threshold of 0.001. After removing redundant sequences from the original dataset, the same number of sequences was sampled as non-redundant training and evaluation datasets for cross-validation.An eCLIP-seq dataset previously generated from the ENCODE3 database by k = 3–6) representations of nucleotide sequences obtained from a human reference genome, GRCh38.p13. Once the CLS and SEP tokens were appended to the input k-mers, each token was embedded into real vectors with 768 dimensions. The model was pretrained with the masked language modeling objective to predict randomly masked tokens using information from other tokens. The model had 12 transformer encoder layers, each of which consisted of 12 self-attention heads and utilized the multihead self-attention mechanism . The models were trained on four NVIDIA Tesla V100 GPUs (128 GB memory). The training of one RBP model using 19 200 samples took <10 min. After fine-tuning, the model performance was measured using independent test sets using the area under the receiver operating characteristic curve (AUROC).Upon fine-tuning, the parameters of the model were initialized with those of DNABERT . SubsequThe following four existing models were implemented as baselines: GraphProt, iDeepS, HOCNNLB and DeepCLIP. GraphProt is an SVM-based model that converts RNA sequences and their estimated secondary structures into graph representations and predicts RBP-binding sites  is an indicator that returns 1 if the property exists in the nth sequence or 0 otherwise; L indicates the number of sequence tokens in the nth sequence, and L + 2 is the number of all tokens, including CLS and SEP. Intuitively, f.When conditioned by an input sequence, each head emits a set of attention weights g is an indicator that returns 1 if the property exists in the jth token of the nth sequence in the lower layer and 0 otherwise. Note that attention weights to CLS and SEP were not considered during the token-level analysis, and the token length L was used. Here, g.For token-level analysis, attention weights to the token of interest were computed at each head using the following equation:We first examined whether attention weights reflect transcript region types, including the 5′UTR, 3′UTR, intron and CDS. Region-type annotations were downloaded from the Ensembl database  using LinearPartition  was compP-value of 0.005 to declare statistically significant enrichment in the positive samples. Finally, similar motif candidates were merged using pair-wise alignment, and the resulting motifs with the highest number of instances were selected. Selected motifs were compared with those detected using an ab initio motif discovery tool, STREME  and BERT-baseline. All models were trained on the same training set, and their performance was measured using an independent test set over 154 RBPs. BERT-RBP resulted in an average AUROC of 0.786, which was higher than that of any other model . The aveBecause the original DNABERTs were pretrained on 3- to 6-mer representations, we fine-tuned three other models, where each model takes 4- to 6-mer representations as inputs. When the AUROCs of fine-tuned models with different k-mers were compared, all fine-tuned models showed comparable performance to the 3-mer model, again demonstrating the robustness of the two-step training method . The detWhile being a deep learning model, BERT has high interpretability . To validate the extracted motifs, we applied STREME, an  methods . Hence, As our analysis demonstrated the model’s capability to translate transcript region type and RNA secondary structure, DNABERT can potentially be applied to other RNA-related tasks, such as RNA subcellular localization prediction (d is the number of transformer layers, and n is the number of tokens. The syntactic relationship among tokens is another intensely researched topic in BERTology and may incorporate hidden contextual patterns of nucleotide sequences (Although our analysis implied that the fine-tuned model could utilize the information learned by DNABERT, it must be noted that the attention analysis itself does not necessarily provide a comprehensive explanation of how the model processes information (equences . In the In this study, we proposed BERT-RBP, a fine-tuned BERT model for predicting RNA–RBP interactions. Using the eCLIP-seq data of 154 different RBPs, our model outperformed state-of-the-art methods and the baseline BERT model. Attention analysis revealed that BERT-RBP could distinguish both the transcript region type and RNA secondary structure using only sequence information as inputs. The results also inferred that the attention heads of BERT-RBP could either utilize information acquired during DNABERT pretraining or vary the type of information processed when necessary. Thus, this study provides a state-of-the-art tool to predict RNA–RBP interactions and infers that the same method can be applied to other bioinformatics tasks.vbac023_Supplementary_DataClick here for additional data file.