Cancer is one of the leading causes of death worldwide. Human cytomegalovirus (HCMV), a well-studied herpesvirus, has been implicated in malignancies derived from breast, colorectal muscle, brain, and other cancers. Intricate host-virus interactions are responsible for the cascade of events that have the potential to result in the transformed phenotype of normal cells. The HCMV genome contains oncogenes that may initiate these types of cancers, and although the primary HCMV infection is usually asymptomatic, the virus remains in the body in a latent or persistent form. Viral reactivation causes severe health issues in immune-compromised individuals, including cancer patients, organ transplants, and AIDS patients. This review focuses on the immunologic mechanisms and molecular mechanisms of HCMV-induced carcinogenesis, methods of HCMV treatment, and other studies. Studies show that HCMV DNA and virus-specific antibodies are present in many types of cancers, implicating HCMV as an important player in cancer progression. Importantly, many clinical trials have been initiated to exploit HCMV as a therapeutic target for the treatment of cancer, particularly in immunotherapy strategies in the treatment of breast cancer and glioblastoma patients. Taken together, these findings support a link between HCMV infections and cellular growth that develops into cancer. More importantly, HCMV is the leading cause of birth defects in newborns, and infection with HCMV is responsible for abortions in pregnant women. HCMV is a pathogen infecting 70-90% of the world population that remains latent over the lifetime of the host after primary infection and reactivates to cause severe disease in immunocompromised patients. HCMV infection in humans causes the progression and development of several cancers, including breast cancer in women , brain  and coloThe oncogenic potential of HCMV is associated with the presence of a number of oncogenes in its genome, such as IE1, IE2 , US28 M, and UL7in vitro experiments  occurs in all multicellular organisms and is a process that is often evaded in cancer. Apoptosis is an antiviral response that is detrimental to HCMV growth cycle because it kills infected cells and thus prevents persistent HCMV infection. Therefore, HCMV have several strategies to prevent premature or programmed cell death of infected host cells to survive and spread to adjacent cells. HCMV has been reported to prevent apoptosis of cancerous cells, and this anti-apoptotic effect is mediated by IE proteins via both p53-dependent and p53-independent pathways . Moreove3.3Apoptosis is a programmed cell death that may be activated by cell stress (intrinsic pathway) or cytokines (extrinsic pathway). Studies have shown that HCMV encodes a UL36 protein that inhibits caspase-8 activation  or M36 h3.4Cell invasion, migration, and adhesion to the endothelium are very important events that take place during the process of tumor formation, and it has been reported that HCMV infection could have some association with these processes. Important cellular signaling events such as phosphatidylinositol-3 kinase/protein kinase (PI3K/AKT) and mitogen-activated protein kinase (MAPK) pathways, are responsible for cell growth and proliferation and are known for their essential role in tumor formation . Studies3.5Macrophages and other monocytes represent the first line of defense against pathogens and are activated by a T helper-1 (Th-1) type of response via IFN-γ secretion. Macrophages constitute the major reservoir for HCMV, while fibroblasts are the most widely used cell type to experimentally grow HCMV. Macrophage differentiation is inhibited by HCMV in order to replicate and grow optimally in this cell type. An intact ULb’ region has been identified in the genome of HCMV that allows the virus to grow in macrophages, endothelial, and epithelial cells . However44.1Breast cancer is the leading cause of cancer-related deaths in women and impacts over 1.5 million individuals every year . The majin situ hybridization, and PCR, the presence of HCMV antigens was detected in glandular epithelium 17/27 (63%) of normal adult breast tissues (in situ (DCIS) and infiltrating ductal carcinoma (IDC) (HCMV has been found in the breast milk of more than 90% of lactating women that are seropositive for anti-HCMV antibodies  and is c tissues . In thisma (IDC) . The exama (IDC) . A totalma (IDC) . Moreovema (IDC) . A new sma (IDC) . HCMV-DBma (IDC) . The HCMma (IDC) . In thisma (IDC) .In a recent study, it was found that 70 of 146 (47.9%) breast cancer samples were positive for HCMV IE proteins and CMV late antigens were detected in 78/146 (53.4%) samples from breast cancer patients . More im4.2in situ hybridization assays. Using a murine CMV in a syngeneic GBM model, a recent study has reported that CMV markedly increases tumor growth and leads to a significant reduction in overall survival. This study identified that the PDGF-D factor is responsible for the CMV-mediated angiogenesis and tumor growth  is a life-threatening disease that is the third-leading cause of cancer-related deaths. In the past decade, scientists have made many efforts on finding the mechanisms of this disease and developing effective treatments. CRC pathogenesis is associated with various environmental components, such as lack of exercise, high-fat diet, smoking, and the environment in developed countries. However, scientists have recently been attracted to the contribution of microorganisms such as HCMV to CRC pathogenesis. Many studies have confirmed the presence of HCMV DNA in CRC tumor tissues, indicating an unidentified role of HCMV in the development of the CRC . It was 4.4Carcinoma of muscle is rare compared to other tissue cancers like bone, lung, pancreas, and liver. Smooth muscle cells are frequently infected by CMV . Majorly4.5in situ hybridization assays to detect HCMV infection. In another study, HCMV RNA was found in prostate cancer tissues using RNA sequencing methods , which targets viral DNA replication, is the most widely used and is the first-line drug in the clinical treatment of HCMV infection . Up to n5.2siRNA , which has been emerged as a powerful tool in gene therapy, may provide a new way to fight viral infections. siRNAs recognize and bind to the target mRNA and in turn, reduce the expression of protein. It has been suggested that siRNA could be useful for the treatment of various diseases, such as cancer, neurodegeneration diseases, ophthalmic diseases and viral infection . Long-te5.3Immunotherapy has emerged as a new approach for the treatment of HCMV-associated cancer. Antigens related to HCMV have been identified as potential targets in immunotherapy. For example, HCMV-associated proteins such as IE1, pp65, and late antigens could be detected in GBM tumor tissues with high specificity. These findings provide promising methods for HCMV-related cancer immunotherapy. Schuessler et al. developed a cellular immunotherapy method to treat GBM by targeting HCMV antigens in patients via autologous T cell therapy. The results demonstrated that the therapy was safe and promising for the treatment of GBM . A studyIt is indicated that CMV can function as an important immunotherapeutic target in cancer therapy for glioblastoma. Some studies have provided evidence that CMV antigen-pulsed autologous dendritic cells, autologous CMV-specific T cells, CMV-targeting CARs, and peptide vaccines are promising approaches for glioblastoma treatment . It has 5.4The protection against HCMV infection in pregnant women is a result of neutralizing antibodies, anti-gB antibodies, anti-PC antibodies, High avidity antibodies, CD4+ T cells, CD8+ T cells, CD4+, CD45RA+ T cells, and cytokines. Natural HCMV infection generates an effective broad immune response comprising the arms of both the innate immune system and adaptive and cellular immunity. During natural infection, the antibodies and T cells specific for PP65, IE1, IE2, and gB play an important role in inducing the immune response. However, sterile immunity against CMV infection has not yet been achieved. Studies have shown that gH/gL/pUL128-131 pentameric complex antibodies are crucial for protection against HCMV infection .The attenuated Towne vaccine generated by the extensive passages in tissue culture had inadequate efficacy in clinical trials . A two-b5.5CMV uses multiple immune evasion strategies to escape from the host immune system. Natural killer (NK) cells are important components of the innate immune system. CMV inhibits NK cells via the CD94/NKG2 heterodimeric receptor by the host HLA-E pathway . StudiesCMV displays several mechanisms to prevent CD8+ T cell recognition, including inhibition of MHC class I processing and presentation pathways. For example, US2 and US11 degraded MHC class I heavy chains , and US3CMV-encoded US2 inhibits MHC class II processing and presentation by degrading the HLA-DRα and HLA-DMα chains . CMV exp5.6miRNAs are a class of noncoding RNAs of 20-24 nucleotides that post-transcriptionally regulate gene expression by forming the RNA-induced silencing complex (RISC) with an mRNA that possesses a complementary sequence. Many studies have shown that HCMV-encoded miRNAs target viral and cellular mRNAs for viral persistence and productive infection. CMV has been found to express miRNA and these CMV-encoded miRNAs were detected in the plasma , saliva Another interesting miRNA encoded by HCMV is CMV70-3P, which increases GBM CSC stemness. This miRNA can also control the expression of SOX2. The inhibition of CMV70-3P prevents glioma cells from proliferating and forming neurospheres . A list 5.7in vitro and in vivo by designing a new HCMV-infected cell targeting system including a chemokine target molecule conjugated to a therapeutic fusion toxin (named F49A-FTP) serves as the most essential protein for cellular infection by the HCMV virion-containing trimeric complex. PDGFR is essential for the entry of the HCMV containing trimeric complex (gH/gL/gO) but not the pentameric complex (gH/gL/UL128-pUL130-pUL131A) . MoreoveF49A-FTP). The cheF49A-FTP). Apart fF49A-FTP). Compare6® as an add-on therapy in patients with malignant glioblastoma and HCMV infection. This clinical trial was mainly based on the hypothesis that HCMV infection can promote tumor development and inhibit immune responses against the tumor. This ongoing study aimed to investigate whether treatment of CMV infection with the antiviral drug Valganciclovir® affects the clinical outcome of GBM in patients with CMV infection . Some additional studies involving in the use of HCMV as a target for cancer therapy have been completed, as shown on the Clinicaltrials.gov website. One study, entitled “Evaluation of recovery from drug-induced lymphopenia using cytomegalovirus-specific T-cell adoptive transfer”, aimed to evaluate whether vaccinating adult patients with newly diagnosed GBM who were seropositive for CMV using CMV-DCs during recovery from therapeutic TMZ-induced lymphopenia with ALT enhances the T cell response and safety. However, some trials, have been terminated, such as the trial entitled “Administration of CMV-Specific Cytotoxic T cells in Patients with Glioblastoma Multiforme” (NCT01205334) carried out by Baylor College of Medicine and the trial entitled “A Study Using Allogenic-Cytomegalovirus (CMV) Specific Cells for Glioblastoma Multiforme (GBM)” (NCT00990496), due to poor patient accrual rates. These results of clinical trials provide evidence that CMV can be used as a target for the treatment of cancer in spite of the mechanism by which HCMV is associated with cancers, such as GBM still needs to be investigated  and (gB) have been suggested to function as potential immunotherapy targets . Strategstigated .7In recent years, the association between tumor progression and HCMV infection has been investigated. Cancer is a major health problem world-wide and causes millions of deaths every year. The aforementioned studies have shown that the HCMV genome contains oncogenes that have the potential to initiate various cancers. Studies have also confirmed the presence of HCMV-specific DNA and antibodies in tumors, indicating a direct role of HCMV-induced cellular transformation. Recently, several clinical trials have started to exploit HCMV as a therapeutic target for various cancers especially glioblastoma.The key factors that HCMV induces cancer progression are still enigmatic, and concrete evidence is still lacking. There could be many unidentified genes in HCMV genome that contribute to cancer progression in host cells. Certainly, there is a need for investigators to address these gaps in knowledge as well as identify and characterize several unidentified genes to begin to fully understand the role of HCMV in tumor progression. Finally, a model of HCMV infection needs to be developed to study the gene-specific contribution to tumor progression, and the underlying mechanisms and biological pathways need to be elucidated.KG, CY, SH and XG conducted the review and wrote the draft. WZ and PR revised and designed the layout of the review. All authors contributed to the article and approved the submitted version.


Acute coronavirus disease 2019 (COVID-19) presents a wide spectrum of clinical manifestations, from asymptomatic infection to severe pneumonia or multisystemic failure. In addition, nearly 3 years after the pandemic, now it is known that there are persistent forms of COVID-19, known as long-COVID, with long-term effects in different organs and systems. These complications related to SARS-CoV-2 infection, which significantly affect the quality of life of many convalescent patients, are not restricted to severe presentations of COVID-19; hence, many patients with persistent symptoms have never been hospitalized. The mechanisms explaining long COVID are not yet well delimited. Recent findings related to immunity alterations together with inflammation and endothelial damage induced by the virus, along with certain predisposing factors, would favor the development of these complications.Tamayo-Velasco et al. detail in a complete review how the presence of anti-antigen A and B antibodies in group 0 patients confers a protective effect against protein S of the virus, which could open new avenues for prognostic and therapeutic stratification. The presence of a high viral load in some individuals determines the status of persistent viraemia, which has also been shown to be an independent factor associated with bad prognosis in COVID-19  in evaluating PACS-CVS. However, it is important to note that there is a lack of evidence-based recommendations for managing this elusive condition. Nonetheless, CPET should be implemented due to its ability to assess the pathophysiology of exercise limitation.The post-acute sequelae of COVID-19 (PACS) represent a heterogeneous group of symptoms characterized by cardiovascular, general, respiratory, and neuropsychiatric sequelae. PACS can be classified into two categories: PACS cardiovascular disease, characterized by a group of cardiovascular conditions that develop during the chronic phase of the disease, and PACS cardiovascular syndrome (PACS-CVS), which lacks clear evidence of cardiovascular disease . In thisGonzález et al. have found that 32.2% of these patients persisted with respiratory alterations, 10% still had moderate/severe lung diffusion (DLCO) involvement (<60%), and 53.7% had a fibrotic pattern on CT. Moreover, patients had a mean (SD) number of symptoms of 5.7 ± 4.6, and 61.3% met the criteria for post-COVID syndrome at 1 year. Thus, there is a compelling clinical need to identify circulating fibrosis markers in COVID-19 leading to pulmonary pro-fibrotic responses that can identify candidate patients suffering from long-term COVID with respiratory alterations. In this regard, Brusa et al. report another circulating biomarker, known as the Targeting Matrix Metalloproteases Pathway-1 (TIMP-1), which has been associated with disease severity and the systemic inflammatory index, suggesting a promising non-invasive prognostic biomarker for structural respiratory damage in COVID-19 patients.In about 25% of patients with severe COVID-19 disease (WHO Severity Grade 3 and 4), a restrictive ventilatory defect has been revealed. This and other facts justify that a significant percentage of COVID-19 patients present respiratory failure not only during the acute illness of the disease but also chronically, months after overcoming it. SARS coronavirus induces the upregulation of type I collagen , and quick identification is key to being able to treat it early. Beyond the classic diagnostic tests of myocardial injury (electrocardiogram and echocardiogram) and cardiac biomarkers (such as troponin and natriuretic peptides), the identification of new affordable and bedside biomarkers seems essential to identify this potentially fatal situation. Recent studies have evaluated the role of MR-proadrenomedullin (MR-proADM), a novel marker of ED in sepsis and pneumonia  assessing the effect of antiplatelet therapy in adult patients with COVID-19. Based on 23 observational studies, including 87,824 COVID-19 patients, antiplatelet treatment has been found to favor a lower risk of mortality . However, the narrative synthesis of RCTs showed conflicting evidence, which did not support adding antiplatelet therapy to the standard care. This discrepancy seems to suggest that there are subgroups of COVID-19 patients who could benefit from this therapy, while in others such a benefit would not exist. It is necessary to carry out new and larger RCTs that evaluate antiplatelets from an individualized or personalized point of view based on the endotype of the candidate patient. In this respect, biomarkers of TE can be useful. D-dimer has shown to be a robust predictor associated with bad outcomes in COVID-19  or endotheliitis has been demonstrated to play a critical role in COVID-19 acute organ disfunction and may also be related to long-term systemic symptoms. ED favors both inflammatory activation and local coagulation, leading to hypercoagulability states (HS), microthrombosis, and hypoperfusion, more markedly in microcirculation . Due to neumonia , 8. It ioversial . In thisCOVID-19 . InteresMoon present a review addressing gastrointestinal symptoms and describing data on the gut–lung axis, viral transmission to the gut, and its influence on gut mucosa and the microbial community.The association of COVID-19 with prevalent gastrointestinal distress, characterized by the fecal shedding of SARS CoV 2 RNA or persistent antigen presence in the gut, has been scarcely evaluated . In thisWe hope that this Research Topic provides original information to the scientific community on the “hot” topic of long COVID and the medium- and long-term effects of SARS-CoV-2 infection.DA drafted the manuscript. LG and DA critically revised the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.


Pancreatic cancer is a devastating disease with a poor prognosis. Novel chemotherapeutics in pancreatic cancer have shown limited success, illustrating the urgent need for new treatments. Lurbinectedin  received FDA approval in 2020 for metastatic small cell lung cancer on or after platinum-based chemotherapy and is currently undergoing clinical trials in a variety of tumor types. Lurbinectedin stalls and degrades RNA Polymerase II and introduces breaks in DNA, causing subsequent apoptosis. We now demonstrate lurbinectedin’s highly efficient killing of human-derived pancreatic tumor cell lines PANC-1, BxPC-3, and HPAF-II as a single agent. We further demonstrate that a combination of lurbinectedin and irinotecan, a topoisomerase I inhibitor with FDA approval for advanced pancreatic cancer, results in the synergistic killing of pancreatic tumor cells. Western blot analysis of combination therapy indicates an upregulation of γH2AX, a DNA damage marker, and the Chk1/ATR pathway, which is involved in replicative stress and DNA damage response. We further demonstrate that the triple combination between lurbinectedin, irinotecan, and 5-fluorouracil (5-FU) results in a highly efficient killing of tumor cells. Our results are developing insights regarding molecular mechanisms underlying the therapeutic efficacy of a novel combination drug treatment for pancreatic cancer. Pancreatic cancer is a devastating disease with a poor prognosis and has an overall 5-year relative survival rate of 12% . A combiLurbinectedin (ZEPZELCATM), developed by PharmaMar yet frequently distributed and advertised by the U.S. based company Jazz Pharmaceuticals, received Food and Drug Administration (FDA) approval in the United States in June 2020 for adult patients with metastatic small-cell lung cancer with progression of the disease on or after platinum-based chemotherapy . LurbineIrinotecan (IRT), introduced to the clinic in 1998, is a cytotoxic prodrug for SN38, which plays a role in the inhibition topoisomerase I. SN38 stabilizes the complex formed by topoisomerase-I and DNA, inducing single-strand breaks. Eventually, these single-strand breaks yield double-strand breaks due to replication fork collapse, triggering subsequent apoptosis . IRT has5-Fluorouracil (5-FU), first discovered in 1957, is used in a variety of tumor types and has been shown to have many potential mechanisms of antitumor activity ,17. 5-FUInterest in lurbinectedin is rising. The drug is being tested in both preclinical and clinical settings as a single agent or in combination with other chemotherapeutics in a variety of tumor types. The first human phase I study of lurbinectedin began in 2009 and occurred in patients with a variety of advanced solid tumors. The aim of the trial was to evaluate dose-limiting toxicities, and it advocated for 1 h intravenous infusion of lurbinectedin every three weeks in a phase II trial . Since tLurbinectedin in combination with gemcitabine in pancreatic adenocarcinoma is further being explored. It has been demonstrated that lurbinectedin synergizes with gemcitabine in pancreatic adenocarcinoma mouse models, inducing elevated levels of DNA damage and tumor associated macrophage depletion . FurtherWhile lurbinectedin is currently being tested in the clinical setting in combination with a variety of therapeutics commonly used to treat pancreatic ductus adenocarcinoma, such as gemcitabine, there is no current ongoing clinical trial involving lurbinectedin combination therapy with irinotecan or lurbinectedin + irinotecan + 5-FU specifically in pancreatic adenocarcinoma. This is an important combination to analyze given the prevalence of irinotecan and 5-FU in modern treatment regimens for pancreatic cancer. One common baseline criticism of a lurbinectedin + irinotecan synergy is high levels of overlapping toxicity. However, it has been demonstrated that lurbinectedin is highly toxic in SCLC cell lines with very low toxicity in healthy lung epithelial cell line HSAECs, indicating lurbinectedin is rather selective for tumor cells . FurtherThus, it is a possibility that a combination of lurbinectedin + irinotecan or lurbinectedin + irinotecan + 5-FU could be a potent combination therapy in pancreatic adenocarcinoma without being excessively toxic. 5-FU specifically is added to this regimen because it is frequently used in combination with irinotecan in the clinical setting. It was hypothesized that adding lurbinectedin to this proven combination may also allow for each individual drug concentration to decrease substantially, making the toxicity more manageable for patients. Our group further explored the combination of lurbinectedin and irinotecan in a preclinical setting using pancreatic cancer cell lines. We report the synergistic killing of such cells and changes in protein and cytokine expression under single-agent and combination therapies. We further report on the highly efficient killing of pancreatic cancer cells with the triple combination between lurbinectedin, irinotecan, and 5-FU.PANC-1 (CRL-1469), HPAF-II (CRL-1997), and BxPC-3 (CRL-1687) are pancreatic cancer cell lines obtained from the American Type Culture Collection (ATCC). PANC-1 was isolated from the pancreatic duct of a 56-year-old white male with epithelioid carcinoma. HPAF-II was isolated from a 44-year-old white male patient with metastasized pancreatic adenocarcinoma. BxPC-3 was isolated from the pancreas tissue of a 61-year-old female patient with adenocarcinoma. While all three cell lines have different genetic profiles, KRAS was activated in PANC-1 and HPAF-II, whereas BxPC-3 was wild type. TP53 mutations are found in all cell lines . All celLurbinectedin was obtained through MedChem Express . Irinotecan was obtained through Sigma . 5-FU was obtained through Selleck Chemicals . In accordance with the manufacturer’s recommendations, each of the above therapeutics were stored at −20 °C.Cell viability was determined via a CellTiter-Glo (CTG) assay. Approximately 3000 cells were plated per well in a black 96-well plate and left to incubate overnight. After 12–24 h allowing for cells to adhere, cells were treated with lurbinectedin, irinotecan, or 5-FU at increasing concentrations to determine single-drug IC-50s. Drug concentrations ranged from a maximum value to a control, and they were diluted via 1:2 serial dilution. The well plates were re-incubated for 72 h. After the re-incubation period, CTG technology indicated cell viability by the luminescent detection of ATP following cell lysis. Brighter luminescence was directly proportional to higher levels of cell viability. Thus, by quantifying luminescence and normalizing to the control, the percentage of live cells was calculated. IC-50 values at 72 h were determined for the three drugs in the three cell lines .HSA = EA,B,…,N − max, in which EA,B,…,N is the combination effect between drugs A, B, …. N and EA, EB,…, EN are responses to each individual drug [Similarly, combination drug treatments of two drugs were performed via a CellTiter-Glo assay. The experimental setup was virtually identical: approximately 3000 cells were plated per well in a 96-well plate and left to incubate overnight. All three previously mentioned cell lines were used. The following day, allowing time for cells to adhere to the plate, cells were treated with lurbinectedin + irinotecan at increasing drug concentrations such that the leftmost upper corner well was a control (no drug treatment) in every plate. CTG reagent was used to evaluate cell viability by lysing cells and fluorescing based on ATP detection following a 72 h drug incubation. To determine cell viability, luminescence was quantified and normalized to the control. Once the percent viability was quantified, combination index analysis was performed. Combination index analysis was performed via the combination index software SynergyFinder, which calculated synergy scores according to an HSA reference model using the following equation: Sual drug ,32. EsseSimilar to two drug combination analysis, triple combination therapy was measured using CellTiter-Glo technology. Approximately 3000 pancreatic adenocarcinoma cells per well were plated and left to adhere overnight. The following day, cells were treated with lurbinectedin + irinotecan + 5-FU and re-incubated for 72 h. The left half of each plate contained lurbinectedin + irinotecan at varying concentrations with no 5-FU, and the right half of each plate had the same concentrations of lurbinectedin + irinotecan with a constant concentration of 5-FU. 5-FU concentration was varied over different 96-well plates. Three-dimensional tensor plots, with each axis corresponding to a different drug, were generated with the color of each point reflecting cell inhibition. Cell viability was calculated by quantifying the luminescence of each well and normalizing to the control (no drug) well in the top left corner of each plate. HSA synergy scores were calculated using the same equation as for the two drug combination therapies and plotted using a heatmap with the axis corresponding to each individual drug concentration. For differing doses of lurbinectedin, a new HSA heatmap for the irinotecan + 5-FU was generated to demonstrate how lurbinectedin affected the synergism between irinotecan and 5-FU.Western blot analysis was performed to analyze the effects of lurbinectedin, irinotecan, and combination in PANC-1, BxPC-3, and HPAF-II pancreatic cancer cell lines. Western blot analysis demonstrated changing protein expression levels, indicating the molecular mechanisms underlying synergy. Cells were plated at approximately 500,000 cells per well in a 6-well plate. After 12–24 h, giving sufficient time for cells to adhere, the cells were treated with control, single, or combination therapies. After 48 h of incubation, cells were harvested from the well plates using a scraper. Cells were lysed with RIPA buffer treated with a protease and phosphatase inhibitor. Proteins were quantified via a BCA assay using the Pierce BCA Protein Assay Kit obtained from Life Technologies . Cell lysates were run through a 4–12% SDS-PAGE gel electrophoresis at 140–170 V and subsequently transferred to a PVDF membrane. The PVDF membrane was blocked for 30 min with 5% non-fat milk (milk powder diluted in TBST). Primary antibodies were diluted at their respective manufacturer’s suggested Western blot dilution in 5% non-fat milk. Following blocking, membranes were incubated in primary antibodies overnight at 4 °C. Invitrogen Goat anti-Rabbit IgG (HRP #31460) and Goat anti-Mouse IgG (HRP #31430) were used as secondary antibodies and diluted 1:5000 in 5% non-fat milk. Each membrane was probed using ECL with an appropriate loading control, which was either vinculin or actin. Western blot densitometry readings were calculated using Image J to find relative protein expression levels. Each protein of interest was normalized to the loading control in the same column.HPAF-II and BxPC-3 cell lines were plated at a density of 75,000 cells/well in a 24-well plate in 0.5 mL of DMEM media. After 16 h, allowing sufficient time for cells to adhere, cells were treated with no drug, lurbinectedin, irinotecan, or combination and re-incubated for 48 h. Following this incubation, media was harvested, and cytokine levels were analyzed using a Luminex Cytokine Detection Assay Panel.Given the documented low IC-50s of lurbinectedin in small cell lung cancer across the literature, it was hypothesized that lurbinectedin may be an efficient killer of pancreatic tumor cells as well. To examine this, HPAF-II, BxPC-3, and PANC-1 human-derived pancreatic adenocarcinoma cells were cultured .After plating three human-derived pancreatic cancer cell lines , cells wGiven that lurbinectedin and irinotecan are currently in a variety of ongoing clinical trials in many tumor types, it was hypothesized that lurbinectedin and irinotecan may synergize in killing pancreatic adenocarcinoma cells, as irinotecan is frequently used in the clinic in a variety of combination therapies to treat pancreatic cancer.Across all three cell lines, the percent inhibition increased as the two individual drug concentrations increased, with the strongest inhibition corresponding to the bottom-right most corner wells of the 60 wells displayed having purple or black fluorescence . Such loGiven that lurbinectedin is an RNA Polymerase II inhibitor shown to induce subsequent DNA damage and irinotecan is a topoisomerase I inhibitor which further causes DNA damage, it was hypothesized that the mechanism underlying synergism is likely rooted in genotoxicity. To test such a hypothesis, markers of DNA damage and other stress pathways were probed for .Following Western blot analysis, cytokine profiling analysis was performed to better understand the molecular mechanisms underlying synergism. A wide panel of cytokines were probed for, and fold change under different treatment conditions is visualized in After establishing the synergism between lurbinectedin and irinotecan and investigating the molecular mechanisms underlying synergism, a triple combination of lurbinectedin, irinotecan, and 5-FU was examined to see if the presence of lurbinectedin could enhance the synergism between irinotecan and 5-FU, which is a drug combination present in many pancreatic cancer regimens . Given tOverall, our results indicate that lurbinectedin is an efficient killer of human-derived pancreatic cancer cell lines as a single agent and in combination with irinotecan and 5-FU, as the drug induces a variety of changes in protein and cytokine expression as a single agent and in combination with other anticancer drugs. Cytokine profiling and Western blot analysis help reveal the mechanism behind lurbinectedin and irinotecan synergism, which is likely rooted in DNA damage, replicative stress, upregulation of death receptor ligands such as FAS, and a downregulation of Bcl-2.Given the poor prognosis of advanced pancreatic cancer, it is imperative that better treatments are researched and eventually implemented in a clinical setting. In treating patient-derived pancreatic cancer cell lines with lurbinectedin as a single agent, lurbinectedin showed a highly potent killing of such cells A in compAfter establishing lurbinectedin as a potential single-agent killer of pancreatic tumor cells, lurbinectedin was combined with irinotecan to determine if the two drugs acted synergistically in killing pancreatic tumor cells. Such a hypothesis was put forward for a variety of reasons. Currently, lurbinectedin and irinotecan are being combined in a variety of tumor types in clinical trials. Additionally, lurbinectedin and irinotecan both act on DNA and are shown to induce DNA damage and breaks. With one drug that inhibits DNA repair and replication, and another that inhibits transcription, it was hypothesized to be a potent combination in killing pancreatic tumor cells, which heavily rely on replication and transcription relative to normal cells. Furthermore, given that irinotecan is a rather standard drug in many chemotherapeutic regimens to treat pancreatic adenocarcinoma, and lurbinectedin is being tested as a monotherapy in a variety of advanced solid tumors (including pancreatic adenocarcinoma), there is potential for such a combination to have clinical relevance.Our hypothesis was confirmed via two drug CTG cell viability assays and subsequent SynergyFinder analysis . As a byGiven the strength of the synergism between lurbinectedin and irinotecan, Western blot analysis and cytokine profiling analysis were both performed to better understand the mechanisms underlying the synergistic killing of the cancer cells under the individual and combination drug conditions. As hypothesized, combination conditions across multiple cell lines showed an upregulation of γH2AX A,B, a maNormally, the ATR/Chk1 pathway plays a role in protecting the genome from DNA damage and replication stress , and theAdditionally, Bcl-2 is normally a pro-survival protein , and a sCytokine profiling further revealed the mechanisms underlying synergism. Across the cytokine panel, there were a variety of upregulated and downregulated factors under different treatment conditions in BxPC-3 A and HPAFas, a ligand involved in multiple apoptotic pathways , is upreAfter establishing synergism between irinotecan and lurbinectedin and better understanding the mechanisms behind their synergism, it was hypothesized that a triple combination of lurbinectedin, irinotecan, and 5-FU might be a potent combination therapy capable of killing pancreatic tumor cells at relatively low concentrations of each individual drug. Given irinotecan and 5-FU are commonly used together, and lurbinectedin and irinotecan are being explored as a two-drug combination in clinical trials in many tumor types, it was important to test in the preclinical setting if adding lurbinectedin to a regimen containing irinotecan and 5-FU would be feasible. If such a drug combination can be affirmed in the preclinical setting, there could potentially be clinical application for such a triple combination.Given the well-documented antitumor activity of the three chemotherapeutics, it is rather unsurprising that Overall, the data indicate that lurbinectedin monotherapy and combination therapy with irinotecan or irinotecan and 5-FU are potent killers of BxPC-3, PANC-1, and HPAF-II. The two-drug combination seems to rely on DNA damage and replicative stress and an upregulation of cytokines related to apoptotic pathways. The triple combination therapy of lurbinectedin, irinotecan, and 5-FU demonstrates a highly efficient and synergistic killing of pancreatic cancer cells, as lurbinectedin enhances the synergism between 5-FU and irinotecan. Our results are investigating the molecular mechanistic basis underlying the novel synergistic combination in pancreatic adenocarcinoma cells.Future experiments will include mouse models in a tumor growth and mouse survival study to test whether lurbinectedin, irinotecan, and 5-FU monotherapy or combination treatments can reduce tumor size and prolong lifespan. To better understand the mechanism behind lurbinectedin and irinotecan’s synergism, more biomarkers for DNA damage, replicative stress, caspase activation, and apoptosis need to be probed for via Western blots. Future experiments will also include co-culturing cytotoxic CD8+ T-lymphocytes as well as Natural Killer (NK) cells with pancreatic tumor cells under monotherapy and combination therapy conditions to analyze whether the drugs are capable of sensitizing tumor cells to immune killing. Such experiments will provide a more robust analysis of the drug combination and shed further insights into whether the drug combination can modulate the tumor microenvironment. Such work will further support the translation of the combination in a clinical setting.Overall, the data indicate that lurbinectedin monotherapy and combination therapy with irinotecan or irinotecan and 5-FU are potent killers of human-derived pancreatic adenocarcinoma cell lines. The two-drug combination appears to rely on DNA damage and replicative stress and an upregulation of cytokines related to apoptotic pathways. The triple-combination therapy of lurbinectedin, irinotecan, and 5-FU demonstrates a highly efficient and synergistic killing of pancreatic cancer cells, as lurbinectedin enhances the synergism between 5-FU and irinotecan. We expect in the future that this therapeutic approach may be of use for patients with locally advanced or metastatic pancreatic cancer. Our results are developing a novel preclinical synergistic combination in pancreatic cancer with a feasible path to testing such a combination in a clinical setting.


Liquid biopsy has emerged as a promising, non-invasive diagnostic approach in oncology because the analysis of circulating tumor DNA (ctDNA) reflects the precise status of the disease at diagnosis, progression, and response to treatment. DNA methylation profiling is also a potential solution for sensitive and specific detection of many cancers. The combination of both approaches, DNA methylation analysis from ctDNA, provides an extremely useful and minimally invasive tool with high relevance in patients with childhood cancer. Neuroblastoma is an extracranial solid tumor most common in children and responsible for up to 15% of cancer-related deaths. This high death rate has prompted the scientific community to search for new therapeutic targets. DNA methylation also offers a new source for identifying these molecules. However, the limited blood sample size which can be obtained from children with cancer and the fact that ctDNA content may occasionally be diluted by non-tumor cell-free DNA (cfDNA) complicate optimal quantities of material for high-throughput sequencing studies.In this article, we present an improved method for ctDNA methylome studies of blood-derived plasma from high-risk neuroblastoma patients. We assessed the electropherogram profiles of ctDNA-containing samples suitable for methylome studies, using 10 ng of plasma-derived ctDNA from 126 samples of 86 high-risk neuroblastoma patients, and evaluated several bioinformatic approaches to analyze DNA methylation sequencing data.We demonstrated that enzymatic methyl-sequencing (EM-seq) outperformed bisulfite conversion-based method, based on the lower proportion of PCR duplicates and the higher percentage of unique mapping reads, mean coverage, and genome coverage. The analysis of the electropherogram profiles revealed the presence of nucleosomal multimers, and occasionally high molecular weight DNA. We established that 10% content of the mono-nucleosomal peak is sufficient ctDNA for successful detection of copy number variations and methylation profiles. Quantification of mono-nucleosomal peak also showed that samples at diagnosis contained a higher amount of ctDNA than relapse samples.Our results refine the use of electropherogram profiles to optimize sample selection for subsequent high-throughput analysis and support the use of liquid biopsy followed by enzymatic conversion of unmethylated cysteines to assess the methylomes of neuroblastoma patients. MYCN amplification, INRG stage, ploidy status, and 11q aberration) are used to divide patients into three categories: low, moderate, and high  (DNA methylation profiling has become a promising approach for sensitive and specific detection of many cancers. Bisulfite conversion of unmethylated CpGs has been the standard method for methylation profiling. Reduced Representation Bisulfite Sequencing (RRBS) is a good method for DNA methylation studies, covering about 3 million CpG sites, with an affordable cost . Howevergnature) –33.Recent studies with immunoprecipitation of cell-free methylated DNA coupled with next-generation sequencing (cfMeDIP-seq) have shown this an effective approach for the analysis of cfDNA methylome from minute quantities of cfDNA , 35. HowIn the present article, we use ctDNA from high-risk neuroblastoma patients to compare two conversion methods of unmethylated cysteine, bisulfite and enzymatic conversion, for whole methylome sequencing studies. We also analyze the tumor fraction (ctDNA/cfDNA) content at diagnosis and relapse to establish the suitable threshold for optimal analysis of tumor methylome profile. Several key points for the successful methylome study of high-risk neuroblastoma are indicated in our results.A retrospective study was performed in a primary cohort of 86 patients diagnosed with primary high-risk neuroblastoma who underwent treatment between 2007 and 2019. 79 patients were classified in M stage and 7 in L2 stage, with mean age of 42 months at diagnosis. 43 relapsed (the mean time to relapse was 17.5 months), of whom 41 samples were collected. These cases were collected from the archives of Hospital La Fe in Valencia. Clinical characteristics for each patient are summarized in Whole blood samples were collected in PAXgene tubes and kept at room temperature for no more than 24 hours before plasma was collected by centrifugation two consecutive times for 15 minutes at 2000xg. Plasma was stored at -80°C until the moment of use. DNA was isolated from 1 ml of blood-derived plasma with QIAamp Circulating Nucleic Acid Kit (QIAGEN). Quantity and quality of isolated ctDNA was determined by using D1000 ScreenTape assay in Agilent 4200 TapeStation. We have also used this method to quantify DNA, focusing on mono-nucleosomal fragments around 100-200 base pairs, but other larger nucleosome sizes were also considered. The median of ctDNA from diagnosis and relapse samples, collected from 1 ml of blood, was 24.2 ng. Quantity of double-stranded DNA was also determined with Qubit (Thermofisher).This method is based on the conversion of unmethylated cytosine to uracil, while methylated cytosines remained unchanged. The QIAseq Methyl Library Kit (QIAseq) was used to prepare libraries from bisulfite-treated DNA samples for whole genome methylation studies using Illumina platforms. QIAseq Methyl Library kit allows DNA inputs as low as 100 pg to deliver high-quality, high-yield libraries for use with Illumina platforms. Libraries were constructed with 10 ng or 20 ng of DNA, as indicated, following the manufacturer recommendations.NEBNext Enzymatic Methyl-seq (EM-seq) was used for the identification of 5-methylcytosine and 5-hydroxymethylcytosine. 10 or 20 ng starting material of cfDNA, as indicated, were used on a two-step conversion of the cytosines. In the first step TET2 oxidation of 5-methylcytosine and oxidation enhancer of 5-hydroxymethylcytosine provided protection to the modified cytosines from conversion by APOBEC deamination, performed in the second reaction. Ultimately, cytosines are sequenced as thymines and 5-methylcytosines or 5-hydroxymethylcytosines are sequenced as cytosines. The final 8 cycles of PCR library amplification were carried out using the NEB Unique dual index primer pairs (NEBNext multiplex oligos for Illumina).The libraries were sequenced on NovaSeq 6000 (Illumina) in paired-end with a read length of 2x151 bp according to the manufacturer’s protocol for dual indexing. Image analysis, base calling and quality scoring of the run are processed using the manufacturer’s software Real Time Analysis (RTA 3.3.3) and followed by generation of FASTQ sequence files.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE221317).The EM-seq reads were processed using the gemBS pipeline v4.03 using as reference GRCh38. Reads with MAPQ scores < 20 and read pairs mapping to the same start and end points on the genome were filtered out after the alignment step. The first 5 bases from each read were trimmed before the variant and methylation calling step to avoid artifacts due to end repair. For each sample, CpG sites were selected where both bases were called with a Phred score of at least 20, corresponding to an estimated genotype error level of <=1%, and where the total number of reads informative for methylation from both strands combined was >=6. Sites with >500x coverage depth were excluded to avoid centromeric/telomeric repetitive regions. Sequencing depth for the analysis of CNV was calculated using samtools v1.15. Liquidhope cohort data is available at Geo database . Both techniques generated similar amounts of sequencing data with no significant differences, referred to by either total millions of reads . With the EM-seq method, we obtained 99.8% conversion efficiency using this metric, and with QIAseq method we obtained a conversion efficiency of 99%. The comparison of the library yield (Gb generated in the sequencing run) between QIAseq and EM-seq showed similar results  to improve the ratio between ctDNA and cfDNA , contained CNVs as detected by sequencing analysis. In addition, the profile of methylation averaged over 1kb bins for these samples and displayed extended regions of hypo-methylation with blocks of methylation extending over many kb with average methylation below 0.5 . In addition to a rather small mono-nucleosomal peak, normal cfDNA may contain multi-nucleosome peaks with increasing DNA quantities , further supporting that the mono-nucleosomal fragment is an indicator of ctDNA content.Samples containing a mono-nucleosomal peak at around 100-200 base pairs, with or without a smaller peak at di-nucleosomal size and sometimes also at tri-nucleosomal size  of the sequenced methylome in each sample  did not exhibit a significant statistical difference between diagnosis and relapse samples , we evaluated two methods, the QIAseq, based on the standard genome bisulfite conversion, and EM-seq methods, based on two-step enzymatic conversion process. Our comparison between the different techniques indicates that, although conversion efficiency, library size and integrity obtained were optimal in both methodologies, we achieved higher library concentration, five to six times higher, with the EM-seq approach .MYCN amplification at diagnosis had only 3 copies at relapse, with no other genetic or epigenetic alterations) after sequencing analysis. In healthy individuals, cfDNA is very low, averaging 10 to 15 ng per milliliter . In addition, the hypomethylation profile also enabled us to discriminate between tumor and normal cfDNA containing samples. DNA methylation changes are similar in most cells of a tumor but in contrast, the manifestations of gene mutations are more heterogeneous within the same tumor . Thus, tOne interesting observation from our studies is that relapsed samples usually contained lower amounts of ctDNA  per ml of blood than their paired samples at diagnosis. Consistent with this, ctDNA at relapse was more easily masked by normal cfDNA, even though all samples were carefully subjected to the same technical procedures. Although we do not yet have a clear explanation for these findings, one possibility is that high-risk neuroblastoma at relapse is a more disseminated disease associated with less hypoxia. Therefore, hypoxia-associated processes such as apoptosis and necrosis, are also reduced and thus, less ctDNA is released into the bloodstream than at diagnosis. Alternatively, neuroblastoma relapses are more frequently found in bone marrow, which could represent a barrier for liberating tumor DNA to the bloodstream.Collectively, our results support the use of cytosine enzymatic conversion over chemical modification as an optimal method to study ctDNA methylome with as little as 10 ng of DNA and provide a conceptual tool for the electropherogram-based detection of samples with useful ctDNA content for subsequent studies.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/La Fe Research Ethics Committee approved this project, reference number 2018-02-09. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.ET: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing. AJ-R, GP, MG: Collection and/or assembly of data, data analysis and interpretation. VC, AC: Conception and design data analysis and interpretation, manuscript writing. SH: Conception and design data analysis and interpretation, manuscript writing. JF: Financial support, conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, coordination. All authors contributed to the article and approved the submitted version.


Sarcopenia prevalence depends on the definition, and ethnicity must be considered when setting reference values. However, there is no specific cut-off for sarcopenia in Saudi women. Accordingly, we aimed to establish a cut-off value for sarcopenia in Saudi women. We determined the prevalence of sarcopenia in terms of low handgrip strength (HGS) in postmenopausal women using the EWGSOP2 value, redefined a specific cut-off for low HGS derived from Saudi premenopausal women, re-determined the prevalence of low HGS using the new cut-off, and analyzed the proportion of low HGS in women with obesity compared to those without obesity. Following EWGSOP2 guidelines, we defined probable sarcopenia and set new HGS values. We assessed HGS and body composition in 134 pre/postmenopausal women. Probable sarcopenia prevalence was calculated using EWGSOP2’s HGS of 16 kg and new cut-offs from young premenopausal women without obesity. HGS 10 and 8 kg cut-offs were calculated from premenopausal Saudi women’s mean −2 SDs and mean −2.5 SDs. Using the HGS 16 kg cut-off, sarcopenia prevalence was 44% in postmenopausal and 33.89% in premenopausal women. Applying the new HGS 10 kg and 8 kg cut-offs, the prevalence was 9.33% and 4%, respectively, in postmenopausal and 5% and 3.40%, respectively, in premenopausal women. Women with obesity had a higher proportion of low HGS across all cut-offs. We suggest that EWGSOP2 cut-offs may not be adaptable for Saudi women. Considering body composition differences between Saudis and Caucasians, our proposed HGS cut-offs appear more relevant. Sarcopenia is a progressive skeletal muscle syndrome that accelerates muscle mass and function loss, increasing mortality risk, falls, fractures, and physical disability . RosenbeHandgrip strength (HGS) is a valuable muscle strength index recommended to diagnose sarcopenia . As muscSarcopenia-related cut-off values might differ by the demographics, especially among height and muscle strength-based metrics. EWGSOP2 pointed out that it may be better to employ population-specific cut-offs if available. As the prevalence of low HGS changes along with the HGS cut-off, for instance, the HGS cut-offs suggested in EWGSOP1 (30/20 kg) are significantly higher than the EWGSOP2-recommended HGS cut-offs (27/16 kg) for detecting probable sarcopenia. Accordingly, EWGOSP2 suggested using HGS cut-offs lower than 2.5 standard deviations from the mean value of the HGS of the young adult in a specific population . We used bioelectrical impedance analysis to estimate the lean mass of legs and arms.The surrogate sarcopenia index for muscle mass, defined as appendicular skeletal muscle mass (ASM) , was estWe measured anthropometrics and body composition following the study protocol’s standardized operating procedures.2) and categorized using the World Health Organization standards (WHO)  and [mean −2.5 SDs] from the EGWSOP2 consensus. An HGS of 10 kg is the cut-off point proposed to identify probable sarcopenia in Saudi women, and an HGS of 8 kg is a conservative measure for this purpose. These values are lower than the cut-off values found in other populations, such as Turkey , Italy , Spain , and BriIn a study similar to ours, Bhat et al. reported HGS cut-offs in Turkey using EWGSOP2’s method to determine low HGS as mean −2.5 SDs of young Turkish adults as a reference group and compared these Turkish-specific values to other published population-specific cut-offs. The Turkish study included 114 females, and the mean HGS of young Turkish women was 33.10 SD ± 5.30) kg, and the HGS was 22.5 kg and 19.8 kg when the HGS cut-off was calculated as mean minus 2 SDs and mean minus 2.5 SDs of the young reference women, respectively . Individ kg, and The handgrip strength test is vital in determining frailty and predicting all-cause mortality, cardiovascular mortality, and morbidity. It is quick, inexpensive, and simple to conduct, making it a promising tool for stratifying an individual’s risk of disease. However, published reference ranges for handgrip strength are mostly based on Caucasian individuals, with limited data on non-Caucasian populations ,30. Several methodologies have been employed in the literature to estimate handgrip strength cut-offs for identifying low strength. Studies conducted in Finland, Italy, and Turkey have established an HGS threshold, derived from receiver operating characteristic (ROC) analysis, that predicts mobility limitations or slow gait speed ,31,32. In the literature, it has been conventional to employ statistical methods, such as dichotomization, to categorize patients into low and normal appendicular skeletal muscle mass index (ASMI) based on various cut-off values. However, this method may obscure true dose–response relationships, and limitations in analyzing these variables must be addressed in future sarcopenia studies . ConsequAn Italian epidemiological study investigated the risk factors for mobility and disability related to aging in Tuscany . The stuIn this study, postmenopausal women had weaker handgrip strength than premenopausal women, suggesting a relationship between menopause and muscle strength. These observations support previous investigations indicating that the transition to menopause increases the risk of sarcopenia ,35. MuscMoreover, menopause at a young age may exacerbate the reduction in muscle strength since women are exposed to hypoestrogenism for an extended period. Four cross-sectional studies involving over 10,000 women have been systematically reviewed to examine the impact of physical function and menopause. The studies found that premature menopause before the age of 40 and early menopause before the age of 45 are associated with weaker grip strength, but the likelihood of functional decline decreases in menopause after age 50 . FurtherIn Hispanic women, menopause before the age of 45 significantly increases the risk of developing sarcopenia, and the prevalence of those with low muscle strength evaluated by handgrip strength increases from 7.1% in women in their 40s to 79.4% in those over 80. Similarly, physical performance decreases from 0.5% in the younger group to 60.5% in the older group. The risk of sarcopenia rises sharply from 6.7% in Hispanic younger women to 58.1% in older women. Meanwhile, frailty affects less than 1% of Hispanic women under 60, rising to 39.5% in those over 80 .An interesting investigation of Mexican-American women reported that differences between pre- and postmenopausal women in grip strength become insignificant after age adjustment . HoweverDue to the high prevalence of obesity among Saudi women, we compared the prevalence of probable sarcopenia between those with and without obesity. Using the EGSWOP2 cut-off of 16 kg, we discovered that probable sarcopenia was higher in the obesity group. Similarly, the new Saudi-specific cut-off of 10 kg showed a higher prevalence in the obesity group. However, when a more conservative cut-off of 8 kg was employed, the rates of probable sarcopenia were comparable between the obesity and non-obesity groups.The significant differences between premenopausal and postmenopausal women in handgrip strength in our sample, with insignificant differences in muscle mass, could be attributed to several reasons, including bone mineral density, obesity, and menopause-related hormonal alterations. Evidence from the literature shows a positive correlation between handgrip strength and bone mineral density in premenopausal women , while nAlthough muscle mass strongly predicts handgrip strength , our anaObesity is an independent risk factor for decreased muscle strength , linked Losing ovarian hormones during menopause is associated with changes in adipose tissue distribution from subcutaneous to visceral regions .ConsisteSarcopenic obesity is a medical condition that occurs when excess body adiposity and sarcopenia coexist . The croSex-specific differences in the development and progression of sarcopenia have been observed in older adults, with differences in the hormonal and cytokine/chemokine changes that occur . In womeAfter menopause, adipose tissue undergoes phenotypic changes due to metabolic inefficiencies in subcutaneous and visceral adipose tissue . AbildgaThe infiltration of fat tissue into muscle mass can result in micro changes in muscle composition and function, referred to as “muscle quality” ,64. ThesPremenopausal and postmenopausal women differ in age, making it challenging to separate the effects of aging from those of menopause. As noted in our previous study, age strongly predicts increased cardiovascular risk among women . The midSarcopenic obesity is not limited to older adults and can affect younger adults and middle-aged people with obesity , particuWhen determining diagnostic cut-offs for any given measure, it is essential to consider how they relate to the measured parameter’s consequences. In our study, we explored the trend of handgrip strength in Saudi women, proposed cut-offs, and recommended using population-specific values when available. However, more research is needed to examine the proposed cut-offs concerning sarcopenia-related outcomes through long-term studies to confirm their usefulness as a marker of adverse consequences.This study is the first to provide specific cut-offs for low handgrip strength in Saudi women to diagnose sarcopenia based on muscle strength. Our novel approach considers menopausal status. We focused on handgrip strength instead of muscle mass, as the latter is more closely related to functional decline and adverse outcomes. The primary outcomes were evaluated using widely used and validated measurements, including handgrip strength and bioelectrical impedance analysis, to assess muscle strength, appendicular skeletal muscle, and body composition. An additional advantage of the cut-off points is their practicality in clinical settings, as they are simple and easy to apply. We also developed new cut-offs using the EGWSOP2 algorithm, which fully meets the EGWSOP2 consensus on the definition and diagnosis of sarcopenia. This study’s cross-sectional design and small sample size are limitations that must be considered when interpreting the results, as they affect the study’s generalizability. Since the criteria for sarcopenia were derived from a small number of participants, further research with a larger cohort of Saudi non-sarcopenic young women without obesity is required to extend the findings to Saudi women. Our recommendation for future research is to assess the habitual physical activity level, which is crucial in determining muscle mass and strength. It would also provide valuable insight into Saudi women’s level of physical activity and whether they are as inactive as women in the United States and Europe.The discrepancy in sarcopenia rates based on different cut-off values among Saudi postmenopausal women highlights the importance of using ethnic cut-points in clinical practice. We evaluated the impact of using the EWGSOP2 cut-off versus population-specific cut-offs on estimating the prevalence of probable sarcopenia, characterized by low handgrip strength, in Saudi women. Our study showed that the EWGSOP2 cut-off for low HGS diagnosis to detect sarcopenia in women is not adaptable to Saudi postmenopausal women. Although HGS was lower in Saudi postmenopausal women, Saudi premenopausal women were still diagnosed with low HGS using the current cut-offs. Developing new population-specific cut-offs from Saudi women may be clinically relevant and more adapted. However, before this new criterion is implemented in the clinical routine, future studies must fully comprehend these findings’ consequences and determine whether they correlate with declines in physical function.


Locally advanced or metastatic cholangiocarcinoma is an aggressive carcinoma with a dismal prognosis. For the first-line treatment of locally advanced or metastatic cholangiocarcinoma, cisplatin/gemcitabine has been the standard of care for more than 10 years. Its combination with the immune checkpoint inhibitor durvalumab resulted in an efficiency improvement in the phase III setting. Regarding the use of chemotherapy in the second line, positive phase III data could only be generated for FOLFOX. The evidence base for nanoliposomal irinotecan  plus 5-fluorouracil (5-FU) and leucovorin (LV) is contradictory. After the failure of first-line treatment, targeted therapies can be offered if the molecular targets microsatellite instability-high (MSI-H), IDH1, FGFR2, BRAF V600E, and NTRK are detected. These targeted agents are generally preferable to second-line chemotherapy. Broad molecular testing should be performed, preferably from tumor tissue, at the initiation of first-line therapy to timely identify potential molecular targets. The term “cholangiocarcinoma” (CCA) comprises a group of heterogeneous malignant tumors arising at any point of the biliary tree. Three subtypes of CCA are distinguished according to the anatomical site of origin: intrahepatic, perihilar, and distal , 2. Althth October 2022, to reach a consensus on the systemic treatment of non-resectable, locally advanced, or metastatic CCA.At present, the systemic treatment landscape is expanding, while the currently available options leave room for discussion regarding the ideal choice and sequence of therapies. Therefore, Austrian experts in the field of medical oncology and liver surgery convened on 9The phase III ABC-02 trial published in 2010 established cisplatin plus gemcitabine as the first-line treatment standard in patients with advanced biliary tract cancer . CompareThis long-lasting standard regimen was recently augmented with the anti-PD-L1 antibody durvalumab based on the phase III TOPAZ-1 trial that explored its addition . The OS Due to the long-term effect observed in TOPAZ-1, durvalumab plus cisplatin/gemcitabine has been endowed with a 4-point score according to the ESMO-Magnitude of Clinical Benefit Scale (MCBS) . HoweverAnother factor favoring the immunotherapy-based approach is the possibility of durvalumab maintenance after discontinuation of the cisplatin/gemcitabine backbone. This offers increased tolerability compared to continued administration of the platinum-based regimen, whose prolonged use inevitably evokes complications such as neuropathy. In the TOPAZ-1 study, durvalumab use did not add to the overall toxicity, and the rates of grade 3/4 adverse events were similar across the 2 treatment arms . The ESMAll patients should be re-evaluated regarding potential surgical or locally ablative interventions at regular intervals. It is important for tumor boards to include surgeons specialized in liver surgery, particularly for the assessment of resectability, and to repeat multidisciplinary team discussions 2–3 months after treatment initiation.The addition of durvalumab to first-line cisplatin/gemcitabine should be considered in patients who can be assumed to experience long-term overall survival benefits, that is, individuals with ECOG performance status scores of 0 or 1 who are eligible for doublet chemotherapy and have no contraindications to immune checkpoint inhibition.In patients who have at least achieved disease stabilization with cisplatin/gemcitabine plus durvalumab, durvalumab can be continued as single-agent maintenance therapy after 6 months  of combined treatment.The reinduction of cisplatin/gemcitabine can be considered upon progression after a chemotherapy break of at least 6 months.In patients with locally advanced disease who are candidates for tumor resection, response evaluation to achieve resectability is recommended at 2-month intervals. Restaging in the metastatic setting, on the other hand, should be performed every 10–12 weeks.In the pretreatment setting, the ABC-06 trial is the only positive phase III study conducted to date . ABC-06 The Korean phase IIB NIFTY trial established nanoliposomal irinotecan  plus fluorouracil (5-FU) and leucovorin (LV) as an alternative second-line regimen . Here, pThese findings notwithstanding, nal-IRI plus 5-FU/LV is generally preferred over FOLFOX in Austrian centers and has demonstrated activity in clinical practice . Given tIn cases where nal-IRI cannot be used, palliative therapy with lenvatinib combined with pembrolizumab or regorafenib may be considered for patients with ECOG 0-1 , 22.Second-line administration of FOLFOX is recommended in patients without targetable driver mutations based on phase III evidence.Nal-IRI plus 5-FU/LV represents an alternative option despite controversial phase II data. Early response evaluation after 2 months is recommended.The treatment selection should be based on factors such as performance status and toxicities of previous therapies .FGFR2 inhibitor pemigatinib has been approved for use in pretreated patients with FGFR2 fusions or rearrangements based on the phase II FIGHT-202 trial, which showed an overall response rate of 37% and a disease control rate of 82%  and OS  (In patients with somatic  0.0001) , 25. Dis 0.0001) .BRAF inhibitor dabrafenib and the MEK inhibitor trametinib in 33 patients with BRAFV600E-mutated biliary tract cancer  or mismatch-repair-deficient biliary cancer.NTRK-positive CCA can be treated with the NTRK inhibitors larotrectinib or entrectinib, which have received tumor-agnostic approval for advanced solid tumors harboring NTRK fusions.Patients with IDH1 mutations, FGFR2 fusions, MSI-H, NTRK fusions, BRAFV600E mutations and ERBB2 (HER2) have been classified as level I and three other aberrations as level III according to the most recent ESMO Guideline for Biliary Tract Cancer  . IDH1 mut Cancer . At presBRCA 1/2 testing can identify families with an increased risk of other cancers.Considering the ongoing research efforts in the field of targeted agents, comprehensive testing based on large panels covering driver aberrations beyond those listed by the ESMO Precision Medicine Working Group is encouraged with a view toward patient inclusion in future clinical studies. In addition, Molecular testing of a broad range of targets is recommended prior to the initiation of first-line systemic treatment.Whenever possible, testing should be performed based on tumor tissue.As tissue can be difficult to obtain in the advanced setting, liquid biopsy constitutes a valid alternative. Negative liquid biopsy results do not completely preclude the presence of driver aberrations and should be confirmed if tissue becomes available later.Targeted treatment should be preferred over second-line chemotherapy in patients with ESCAT level I (and II) alterations.Patients with level (II and) III targets, in whom evidence-based regimens have been exhausted, should be discussed by the molecular tumor board.No standard third-line treatments have been defined to date. Oxaliplatin- or irinotecan-based chemotherapy can be administered upon progression following targeted treatment.The recommendations on the systemic treatment of locally advanced or metastatic CCA summarized in this paper mirror the availability of therapies and reimbursement situation in Austria as of autumn 2022 amidst a changing treatment landscape. Data have recently been generated regarding the addition of first-line durvalumab to the chemotherapeutic standard, and despite their limited statistical power, the introduction of immunotherapy represents a potential improvement for certain patients. In the second-line setting, targeted treatment based on potential molecular aberrations is the preferred option.It is strongly recommended to extend molecular testing beyond the established genomic aberrations, as CCA patients—who should be treated at specialized centers as a matter of principle—might get the opportunity to enter clinical trials investigating new compounds. Innovative agents as well as drugs that have already been implemented for other cancers might become accessible over the coming years, thus redefining the current algorithms and taking the systemic treatment of CCA patients to the next level. For patients without druggable targets, FOLFOX is a potential second-line treatment option with a high level of evidence based on a positive phase III clinical trial . The useThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Increased recognition of the development of chronic pulmonary hypertension (cPH) in preterm infants with chronic lung disease (CLD) has prompted enhanced monitoring for the identification of different phenotypes.All newborns consulted for oxygen/respiratory support dependency (CLD assessment) from January 2018 to December 2021 were included. TnECHO and LUS screening for cPH-CLD were performed at 36 weeks postmenstrual age. Cases of cPH related to increased pulmonary blood flow (cPH-IPBF) were referred to Pediatric Cardiology. The objective of the study was to identify all cases of cPH (cPH-CLD/IPBF) in the CLD patients screened and to compare outcomes. Following a standardized algorithm, cPH-CLD patients were treated with diuretics; ultrasounds taken before and after treatment were analyzed.Seventy-two patients with CLD were screened. Twenty-two (30%) had cPH-CLD, and nine (12%) had cPH-IPBF. cPH infants underwent more days of mechanical ventilation, were more likely to have retinopathy of prematurity, and showed increased mortality. The LUS pattern observed in the 72 CLD patients consisted of a thickened pleural line and a B-line interstitial heterogeneous pattern; 29% of patients were found to have lung consolidations. After diuretic therapy, step-down in respiratory support occurred in 59% of neonates with cPH-CLD. A decrease in respiratory rate (RR), right ventricular output (RVO), markers of pulmonary vascular resistance (PVR), and B-line pattern was observed. In tissue Doppler imaging, biventricular diastolic function was found to be modified after diuretics.CLD infants with cPH showed increased morbidity and mortality. In cPH-CLD patients, a decrease in RR and step-down in respiratory support was observed after diuretic treatment. Follow-up ultrasound showed a decrease in RVO, markers of PVR, and B-lines. Chronic lung disease (CLD) is the most common respiratory morbidity in surviving preterm newborns. It is a multifactorial disease, and arises as a consequence of factors associated with immaturity of the airway, decreased alveolarization, and decreased lung growth (both airway and pulmonary vasculature) .Reduced vessel density, abnormal vasculature, and vasoreactivity in infants with CLD set the pattern for the development of chronic pulmonary hypertension (cPH). This complication increases with the severity of the CLD and is associated with higher mortality and respiratory and neurodevelopmental morbidity . In infaAlthough cardiac catheterization is the gold standard for diagnosis of pulmonary hypertension, this procedure is invasive, with a risk of complications due to low pulmonary reserve, and is not feasible in developing countries. For this reason, ultrasound has become the most accessible and feasible tool to characterize and follow different phenotypes for physiology-based management .Significant variability exists in clinical practice regarding screening and treatment. Pulmonary edema and right ventricular (RV) congestion play a significant role in the pathophysiology of respiratory symptoms in patients with cPH. Aside from other markers of pulmonary hypertension (which might be variable), a flat interventricular septum in systole with RV dilatation is used to define cPH-CLD. Diuretics have been used to treat alveolar and interstitial edema in neonates, improving RV loading and relieving symptoms .Jain and collaborators at Mount Sinai Hospital in Toronto developed an algorithm in 2014 for screening at-risk infants at 36 weeks postmenstrual age (PMA), where diuretic therapy was the first-line management approach. They used RV dilatation as a therapeutic threshold; this resulted in symptomatic improvement, disease stabilization, and postdischarge outcomes comparable to those of infants with CLD without cPH .The location of this study was a Pediatric Tertiary Level Referral NICU in Mexico City with a Targeted neonatal Echocardiography (TnECHO) and Point-of-Care Lung Ultrasound (LUS) program. Our primary objective was to identify all cases of cPH among the CLD patients screened and to compare the in-hospital outcomes of patients with CLD only vs. cPH [including cPH-CLD and increased pulmonary blood flow from ASD/VSD (cPH-IPBF)]. The secondary objectives were to characterize LUS findings in the overall population and to evaluate the results of diuretic therapy (through TnECHO and LUS) in cPH-CLD using a standardized algorithm published in a high-income country.This retrospective study was approved by the Institutional Research Ethics committee REB HIM 2021-043, and the requirement for informed consent was waived. The study was conducted from January 2018 to December 2021, and included all infants born at <32 weeks of gestational age (GA) with CLD, defined as oxygen/respiratory support dependence at ≥36 weeks PMA in accordance with the National Institute of Child Health and Human Development consensus definition  with a 9 MHz phased array transducer and a 14 MHz hockey stick probe; during 2020 and 2021, this was a Vivid™ E90  with a 12 MHz phased array transducer and an 18 MHz hockey stick probe.A complete comprehensive echocardiogram was performed in accordance with the 2011 practice guidelines on targeted neonatal echocardiography in the neonatal intensive care unit . Tissue For LUS, six regions of the anterior and lateral thorax were explored with 6 s clips from medial to lateral longitudinal scans . Patients were in a supine position during imaging, so posterior regions were not universally explored . A thick(a)Critical: white lung with coalescent B-lines that obscure A-lines completely; no spared areas.(b)Severe: 25% spared areas.(c)Moderate: 50% spared areas.(d)Mild: 75% spared areas.Following the recommendations of Jain and collaborators, diuretics were started after cPH-CLD diagnosis. First-line therapy consisted of furosemide 1 mg/kg/dose twice daily if administered intravenously or 2 mg/kg/dose twice daily if administered orally for 5–7 days; this was switched to hydrochlorothiazide and spironolactone if administration of the diuretic induced improvement in respiratory symptoms. Pulmonary vasodilators (sildenafil citrate at 0.5–1.0 mg/kg/dose three times daily) were reserved for cases with worsening clinical symptoms or echocardiography features of cPH despite diuretic therapy  after diuretic commencement. Respiratory support and step-down were dependent on the attending neonatology team, with a saturation target range of 90%–94%.Demographic data, management details, and in-hospital outcomes were collected from physical health records. The latest electrolytes taken before treatment and earliest taken after treatment were collected. The radiologic record was reviewed in search of nephrocalcinosis.χ2 test.Statistical analysis was performed using SPSS Version 25.0 (IBM Corp.). Using normality tests for the quantitative variables, any nonparametrically distributed variables were identified; in these cases, data are expressed in the form of medians and interquartile ranges. Data on electrolytes formed a parametric distribution and are reported in the form of averages and standard deviations. The qualitative variables are expressed as absolute numbers and percentages. Bivariate statistics were performed, using the variable of cPH (present vs. absent) as a comparison group the T test (parametric distribution) or the U Mann Whitney test (non-parametric distribution) were used. Median differences before and after diuretic treatment were examined using the Wilcoxon signed-rank test (quantitative variables). Septal flattening  before and after treatment was compared using a linear Seventy-two patients with CLD with a median [interquartile range] gestational age of 29  weeks were screened at 36  weeks PMA. This was a high-risk referral population with deficient prenatal follow-up and with a low rate of prenatal steroids (44.5%). Eighty percent of patients had received surfactant treatment, 68% had early-onset sepsis, and 54% had a healthcare-associated infection. Forty-four percent had a patent ductus arteriosus, with 12% requiring surgical closure. Eighteen percent had severe intraventricular hemorrhage and necrotizing enterocolitis. Ten percent developed retinopathy of prematurity (ROP). Patients with cPH-CLD/cPH-IPBF underwent more days of mechanical ventilation, were more likely to develop ROP, and showed increased mortality before discharge . Except Twenty-two patients (30%) had cPH-CLD and were treated according to the algorithm , 7, and Mortality in our cohort was 9.7%. Fifty-six patients (78%) were discharged with oxygen, and nine (13%) were discharged without oxygen. LUS findings of the 72 CLD patients studied showed a thick pleural line of 0.87  mm and a B-Line interstitial pneumogenic pattern (BLIP). Twenty-one patients showed lung consolidations (29%), which were reported to the attending team. In twelve patients (16%), a superimposed infectious process was integrated after infectious workup .e′ velocity from the left and right ventricular free wall. The right ventricular end-diastolic diameter decreased, although the difference did not reach statistical significance. The incidence of flat septum in systole improved from being universal to an incidence of 32% (linear χ2 test: p < 0.001). Disease progression treated with sildenafil occurred in one case. No electrolyte imbalance was found in the patient charts, and there were no reports of nephrocalcinosis in their radiological archives.Twenty-two patients with cPH-CLD were treated with diuretics (cPH-IPBF was excluded as follow-up was not carried out by the TnECHO team). At the commencement of treatment, three patients were on mechanical ventilation (14%), seven on nasal CPAP (32%), two on a high-flow nasal cannula (9%), and 10 on a low-flow nasal cannula (45%). In the basal LUS, 59% showed moderate edema  and 32% showed severe edema (25% spared areas). After diuretic therapy, a step-down in respiratory support occurred in 59% of CLD-cPH neonates. Two patients remained on mechanical ventilation (9%), two on nasal CPAP (9%), 10 on a low-flow nasal cannula (45%), one on a high-flow nasal cannula (5%), and seven on room air/intermittent oxygen (32%). Subjective BLIP count improved after diuretic therapy, increasing the proportion of mild cases from 9% to 64%; the proportion of moderate cases dropped from 59% to 18%, and the proportion of severe cases dropped from 32% to 18%. With the use of a standardized algorithm, 72 infants were screened as part of our study, identifying 43% with cPH ; this group showed increased morbidity with more days of mechanical ventilation, a higher incidence of ROP, and increased mortality before discharge. This is concordant with the literature, which shows that around 20% of patients with CLD have cPH; this probability increases with moderate-to-severe CLD (30%–50%), and the condition increases the risk of mortality . MouraniTwelve percent of patients screened were found to have significant shunts (cPH-IPBF). These patients were also treated with diuretics, but were followed up by Pediatric Cardiology. In a large study, Kumar and collaborators showed that the presence of an ASD was associated with an increased probability of developing CLD . In anotSavoia et al. published a report on a cohort of 23 preterm newborns where CLD follow-up took the form of echocardiography performed by neonatologists during intensive care and in an outpatient clinic. Excellent inter-rater agreement with the cardiology department was found . Our hosPAAT, or PAAT indexed to RVET , is a noninvasive measure of vascular compliance, resistance, and pressure. Levy and collaborators compared  echocardiography-derived PAAT against right heart catheterization (RHC)-derived systolic and mean pulmonary artery pressure, PVR index, and compliance in a cohort of 75 children. PAAT inversely correlates with RHC-measured pulmonary pressure and directly correlates with pulmonary arterial compliance in children . PAAT ine′ velocity was observed, probably indicative of an enhancement in ventricular relaxation, since this is less sensitive to loading conditions than pulsed Doppler , mainly in early disease , which can contribute to step-down in respiratory management, and we were unable to control for this variable. A prospective evaluation of diuretic administration in this population is needed. In future studies, patient safety needs to be addressed. Although we did not document any complications of diuretic use, the study was not designed to identify these, and laboratory evaluation depended on the attending team. A strength of the study is that it provides a real-world depiction of a standardized protocol for the characterization of different clinical phenotypes of CLD in a developing country that describes favorable outcomes and serves to highlight the importance of considering a randomized control trial.


We report a case of dolutegravir resistance in KwaZulu-Natal in a 13-year-old male two years after starting dolutegravir. Resistance most likely developed due to poor adherence as a result of psychosocial issues. This case highlights the importance of the role of the family unit in impacting adherence and close monitoring of treatment-experienced patients with virologic failure following switching to dolutegravir-based regimens. Numerous challenges affect HIV-infected children and adolescents, impacting adherence, as highlighted in this case. The benefit of HIV drug resistance genotyping to guide the selection of a suitable regimen was shown – there is a need for evidence-based guidelines of indications for resistance testing on dolutegravir.1 Various studies have demonstrated that DTG-based ART is highly effective at achieving viral suppression in both ART-naïve and -experienced individuals. Dolutegravir has better tolerability, higher genetic barrier to resistance and is available in a fixed-dose combination of tenofovir (TDF), lamivudine (3TC), and DTG for adults and adolescents ≥ 35 kg.1 In South Africa, DTG is currently available as a 50 mg tablet for children ≥ 20 kg.1 We present the first paediatric case of DTG resistance identified in a treatment-experienced integrase strand inhibitor (InSTI)-naïve adolescent in KwaZulu-Natal.South Africa transitioned to dolutegravir (DTG)-based antiretroviral therapy (ART) from 2019.1 but it is unclear if he received ritonavir super-boosting during his tuberculosis treatment to overcome the inducing effect of rifampicin. He was diagnosed with tuberculosis again in 2013, again without documentation that LPV/r was super-boosted with ritonavir during treatment. Since ART initiation, he has never been virally suppressed, despite ongoing enhanced adherence support.Our index case is a 13-year-old male from a rural area in who acquired HIV through vertical transmission. The mother tested negative during antenatal care, but she was not tested at delivery. He was diagnosed with HIV and pulmonary tuberculosis in 2010, started tuberculosis treatment and later on ART. As per the national guidelines, abacavir, 3TC and ritonavir-boosted lopinavir (LPV/r) syrups were initiated1 HIV VL is in log10 copies/mL  and CD4 count is in cells/µL . HIV VL ≥ 1000 copies/mL (≥ 3 log10 copies/mL) is defined as virological failure.1In 2018 HIV drug resistance (DR) genotyping was performed, showing only M184V mutation with no protease resistance mutations, which suggested suboptimal adherence. He was switched to zidovudine (AZT) and 3TC, and was started on LPV/r paediatric tablets. The patient received continuous enhanced adherence counselling by the attending doctor and via the social worker. In January 2020, LPV/r was switched to DTG in an attempt to simplify the regimen and decrease the pill burden; however, he remained on AZT/3TC as he weighed 28 kg. The VL at the time of the switch was 529 copies/mL and hence a HIV DR test was not performed. However, he remained unsuppressed and admitted to often forgetting his medication as he lacked a treatment supporter.There were major social issues impacting adherence; he is the product of rape by his mother’s older cousin. Being a teen mom, and not having disclosed the rape issue to her mother proved to have affected the patient’s mother, as well as her ability to provide optimum care for her child. She was the primary caregiver living with the maternal grandmother and was responsible for bringing him for his hospital appointments, but would sometimes not be available to bring him, and occasionally the patient’s biological father would accompany the patient to hospital but identified himself as the patient’s uncle. In 2017, the father became ill, and the grandmother (not knowing the issues around the rape) took him into their home, resulting in family conflict and missed hospital appointments. In 2019, both the patient’s biological parents died due to AIDS-related complications. Upon discovering for the first time at her daughter’s funeral that her nephew had raped her daughter and infected her with HIV, the patient’s grandmother struggled to accept the situation. As a result, the grandmother avoided involving herself in the healthcare of her grandson including supervising him when taking treatment, or accompanying him to hospital, making addressing the adherence issues and disclosure difficult, requiring a home visit by the social workers.Due to poor adherence and patient not suppressing for two years on the DTG-based regimen, despite enhanced adherence counselling, HIV DR was done in April 2022 which showed M184V again and the development of InSTI mutations , conferring high-level resistance to DTG refer to .10 drop.The third-line regimen commenced in May 2022 was TDF, emtricitabine and ritonavir-boosted darunavir once daily. The viral load done after one month was 473 copies/mL with a 2 logThis case report highlights the complexities and psycho-social factors impacting the management of HIV in children and adolescents. The paediatric population has unique challenges such as disclosure, caregiver issues, challenges with understanding and acceptance of the disease, limited drug options and child-friendly formulations.In this case, family issues with a lack of supervision resulted in suboptimal adherence, which resulted in the emergence of DTG resistance. Due to his weight of 28 kg in 2020, he was not eligible for a simple once-daily fixed-dose combination of TDF, 3TC and DTG, which may have improved adherence and potentially prevented DTG resistance.2 None of the participants on first-line DTG-based ART with treatment failure had DTG resistance mutations, while 4/22 (18%) participants with virologic failure on second-line DTG-based ART had DTG resistance.2 Similarly, in the IMPAACT P1093 study that assessed DTG in treatment-experienced children and adolescents, 8/36 (22%) participants with virologic failure had resistance to DTG.3Most clinical trials have evaluated DTG in adults. The ODYSSEY (Once-daily DTG based ART in Young people vs. Standard therapy) trial done in children and adolescents found that DTG was superior to standard care with lower rates of treatment failure.4 R263K reduces DTG susceptibility two-fold, conferring intermediate resistance, while G118R reduces DTG susceptibility by > 5-fold, also conferring intermediate resistance.4 Other resistance mutations selected by DTG in InSTI-naïve patients include E138K/T, N155H, Q148K, S230R, T66I and H51Y.4 Accumulation of mutations further reduces DTG susceptibility. These reported mutations are similar to those found in our case.The most common mutations selected by DTG in InSTI-naïve patients are reported to be R263K and G118R.5In sub-Saharan Africa, real-world cohorts have shown high viral suppression rates in countries that have transitioned to DTG. Although DTG is hailed as the silver bullet, it must still be recognised that treatment failure and resistance, especially in treatment-experienced patients with suboptimal adherence, can occur. The potential factors contributing to DTG resistance include poor adherence, DTG monotherapy, advanced HIV and opportunistic infections.6 The family unit plays an important role in supporting children and adolescents. Decentralisation of care and other avenues of support such as community based family or youth clubs can also help to improve adherence.An evaluation of the ADVANCE (dolutegravir plus two different prodrugs of tenofovir to treat HIV) and NAMSAL  studies highlighted the impact of social and demographic factors on adherence even on DTG-based ART with younger age associated with reduced adherence.The value of VL monitoring and DR genotyping is crucial in detecting cases of resistance. With increasing transitioning to DTG, population surveillance for DTG resistance must be implemented. There is also a need for the availability of a wide repertoire of drug options that are effective, tolerable and cost-effective with child-friendly formulations.This report describes the first case of DTG resistance identified in the paediatric population in KwaZulu-Natal in a treatment-experienced adolescent. It highlights the impact of family and psychosocial issues on adherence, issues affecting adherence in children and paediatric population , and the need to closely monitor treatment-experienced patients with virologic failure, even on DTG-based regimens. The availability of the abacavir/3TC fixed-dose combination and DTG dispersible tablet will help address some of these issues and improve adherence.