Most Zimbabweans access medical care through tiered health systems. In 2013, HIV care was decentralized to primary care clinics; while oncology care remained centralized. Most persons in Zimbabwe with Kaposi sarcoma (KS) are diagnosed late in their disease, and the prognosis is poor. Little is known about whether educational interventions could improve KS outcomes in these settings.Interventions to improve KS detection and management were evaluated at eight Zimbabwe primary care sites  that provided HIV care. Interventions included a standardized KS clinical evaluation tool, palliative care integration, standardized treatment and improved consultative services. Interventions were implemented between February 2013 and January 2016 using a randomized stepped‐wedge cluster design. Sites were monitored for KS diagnosis rates and KS outcomes, including early diagnosis (T0 vs. T1 tumour stage), participant retention and mortality. Analyses controlled for within‐clinic correlations.p = 0.37). New KS diagnosis rates increased 103% (95% CI: 11–273%), p = 0.02) post‐intervention; although paired with an increased odds of incorrectly diagnosing KS . Post‐intervention, non‐significant decreases in 90‐day return rates  and survival  were estimated.A total of 1102 persons with suspected KS (96% HIV positive) were enrolled: 47% incident (new diagnosis), 20% prevalent (previous diagnosis) and 33% determined as not KS. Early (T0) diagnosis increased post‐intervention, though not significant statistically : 0.66–3.79], KS training interventions at urban and rural Zimbabwe decentralized primary care clinics significantly increased overall and incorrect KS diagnosis rates, but not early KS diagnosis rates, 90‐day return rates or survival. LateIn Zimbabwe, the population is mostly rural and obtains medical care, including human immunodeficiency virus (HIV) diagnosis and ART, through a tiered system of local health centres, district, provincial and central hospitals in which nurses and community health workers are the initial primary healthcare contacts . ZimbabwThe National ART Guidelines were amended in 2016 to reflect the Ministry of Health and Child Care commitment to the Global Fast Track 90‐90‐90 targets by 2020 and for consistency with World Health Organization recommendations that all HIV‐positive persons receive ART. Although nurses overseeing ART are trained on the rollout, indications, effects and toxicities of ART drugs, there is little training on the identification, diagnosis and management of HIV‐related malignancies. Training of healthcare workers in cancer detection, treatment and end‐of‐life care is required to reduce the burden of disease and improve outcomes and survival in low‐ and middle‐income countries .To improve outcomes of AIDS‐KS in Zimbabwe and other African settings, KS screening, diagnosis and treatment recommendations must be integrated into primary HIV care settings. Strategies are needed to increase early‐stage KS diagnosis, when it is most likely to respond to ART alone and thereby reduce the need for expensive, difficult to access and potentially toxic chemotherapy. These strategies must be feasible, simple and sustainable in resource‐constrained primary care settings with high HIV burdens. We developed a package of training interventions focused on KS recognition and improved symptom control targeted at primary HIV care providers. We conducted a community‐based randomized stepped‐wedge cluster trial to ensure the intervention was received by all involved clinics. Using this format, we aimed to identify whether training healthcare workers in AIDS‐KS diagnosis and care improved the health outcomes of people with AIDS‐KS.22.1Study design details are available at ClinicalTrials.gov NCT01764360. The study was approved by the institutional review boards of the Medical Research Council of Zimbabwe, University of Colorado Anschutz Medical Campus and the Joint Parirenyatwa Hospital and University of Zimbabwe College of Health Sciences. Written, informed consent was obtained from all study participants.2.2A package of interventions was developed to improve KS care by providing training in clinical recognition and evaluation of KS, palliative care and symptom control see File . The Int2.3The Intervention Package impact on AIDS‐KS outcomes was evaluated by a stepped‐wedge randomized cluster trial design Figure  13, 14], 1413, 12.4The Intervention Package was implemented at eight primary care sites . Urban sites were located within the metropolitan area of Harare and rural sites within a 200 km radius of Harare. All sites received support for HIV care through the President's Emergency Plan for AIDS Relief (PEPFAR) , provide2.5The intervention order for urban and rural sites was block randomized. Within blocks, sites were then randomized to obtain the order for implementation of the Intervention Package. This design ensured that urban and rural sites had a similar number of weeks in the pre‐intervention and intervention periods. Three of the four urban sites were grouped together in the randomization as they were in close geographic proximity and shared staff and patients.2.6Persons of all ages with suspected or confirmed KS who received care at the participating sites at any time during the study period were asked to participate.2.7The study included a Monitoring Team and Intervention Team. Team membership was mutually exclusive and worked independently. The Monitoring Team visited each site weekly throughout the evaluation period. Monitors were blinded to the site randomization results and to when each site began the intervention period. At site visits, the monitors distributed study forms and trained site staff on form completion and keeping daily logs of the total number of patient visits, new KS diagnoses (incident cases) and patients with previously known KS . They did not provide training in any aspect of KS diagnosis or treatment.The Intervention Team included at least one doctor experienced in palliative and AIDS‐KS patient care and one nurse. At each site, the Intervention Team conducted trainings directed at all levels of primary care providers . Training methods were interactive, case‐based and included photographs of common presentations of KS, and discussion of KS cases with various HIV‐ and KS‐related complications. Trainings emphasized early recognition of the disease, including demonstration of a focused full‐body examination for KS, potential KS mimics, and appropriate management of KS and complications including concomitant opportunistic infections. Healthcare providers were watched and given feedback as they did a KS‐focused clinical examination after a training demonstration. In the first month of the intervention period for each site, training included three 4‐hour sessions; thereafter, training was done monthly for 2–4 hours per session through the end of the study. The Intervention Team also provided training to community health workers and traditional healers on KS recognition and referral for medical treatment and distributed posters and brochures in the community to increase awareness of KS.Prior to the start of training and at subsequent follow‐up trainings, healthcare providers were asked to self‐rate their knowledge and skills in six areas  using a 5‐point Likert scale (ranging from 1—“I have no knowledge or skill” to 5—“I am an expert”).To confirm KS diagnosis, doctors and nurses were trained to do punch biopsies of suspected KS skin lesions, and sites were provided with the necessary supplies for specimen collection, storage and transport to pathology at the University of Zimbabwe College of Health Sciences. Biopsies were read by a histopathologist who had completed the AIDS Clinical Trials Group/AIDS Malignancy Consortium training in KS histopathology . Sites w2.8We hypothesized that the training interventions would lead to more active KS surveillance by primary care providers resulting in increased overall and early‐stage KS diagnoses. Analyses included confirmed AIDS‐KS only. Outcomes measured were the proportion of confirmed incident (newly diagnosed) KS over time and the proportion of confirmed incident KS identified at stage T0 (T0/(T0 + T1)) with T0 and T1 defined by ACTG criteria . We also2.9t‐test was used to compare self‐ratings of KS knowledge pre‐ and post‐intervention.Non‐linear mixed effects models were used to account for the site, subject and time effects . Time to33.1The Intervention Package was implemented at each site as shown in Figure 3.2p = 0.021), although the frequency of incorrectly diagnosing someone with KS also increased from 19.9% to 34.3% .Across the eight sites, 1102 individuals with suspected KS were identified. The proportion of HIV clinic visits with suspected KS increased during the intervention period at all sites although the size varied by site Figure , includiOf the 520 individuals with confirmed incident KS, 74 were enrolled pre‐intervention and 446 during the intervention period (Table 3.3p = 0.37)  for the first 90 days; aHR = 1.37  for 90–180 days; and aHR = 2.84  after 180 days. Incident KS 1‐year mortality was 37%  with 41% of deaths identified pre‐intervention compared to 28% in the intervention period. Among incident KS cases, the time to death aHR was 1.36  adjusted for age, sex, T stage, time since HIV diagnosis and stratified by tuberculosis and clinic location to maintain proportional hazards , similar to prior studies in Africa , 20, 21,Although the intervention increased the overall rate of KS diagnosis, the rate of incorrectly diagnosing someone with KS also increased. Diagnosis of KS in African primary care settings is difficult as there are several mimicking disorders , 26, disDespite skin biopsy training and the availability of punch needles and other biopsy supplies, only 16% of incident cases were confirmed by histopathology. We did not investigate the reasons for the low uptake of skin biopsies but suspect the cost of sending biopsies to a central pathology laboratory and the need for additional clinic visits to review biopsy results might have been inhibitory. In sub‐Saharan Africa, the positive predictive values of KS diagnosis by clinical examination compared to biopsy are estimated at 59–84%. Of individuals incorrectly diagnosed with KS requiring alternative treatment, up to 16–35% were considered clinically benign, and 5–6% had alternative diagnoses that were life‐threatening , 29, 30.We did not detect statistically significant effects of the intervention on mortality. Overall mortality was high, potentially due to the large proportion of individuals presenting with advanced KS. Studies completed in sub‐Saharan Africa, including one in Zimbabwe, have reported increased mortality risk among individuals with more advanced tumour burden (T1) , 9, 31. There was evidence that the intervention improved retention in care for newly diagnosed AIDS‐KS. The relative rate of return for a second clinic visit increased over time with incremental increases in the aHR measured at 3‐month intervals after starting the intervention, reaching statistical significance after 180 days. Trainings were provided multiple times at each site. Improvements in retention over time may reflect the time required for a sufficient number of site staff to participate in trainings and for the trained staff to garner experience in applying what they learned to their clinical practice. Repetition in training over time may lead to better improvements in knowledge and application of that knowledge in clinical practice.This study had limitations. Since this study focused on determining the effects of healthcare provider training on the early diagnosis and subsequent health outcomes of individual patients, we collected individual participant but not provider data and so we were unable to identify groups of providers who were more accurate in their KS diagnoses. An incorrect KS diagnosis, either false negative or false positive, could have adversely affected clinical outcomes from lack of appropriate treatment or toxicities from unnecessary treatments. The high mortality does not suggest a high rate of misdiagnosis of KS for otherwise benign conditions. However, if participants had other life‐threatening conditions that were misdiagnosed as KS, incident mortality would have been overestimated. Although including both rural and urban clinics increased the generalizability of our results to AIDS‐KS in Africa, it is important to note that participants had to present to a participating site potentially excluding individuals with more severe KS or those unable to present to a clinic because of physical or monetary barriers, likely leading to an underestimate of T1 stage presentation and overall KS mortality. Our study was also insufficiently powered to detect modest effects on T0 diagnosis (OR<1·89) and on mortality and we cannot exclude the possibility that the intervention had effects smaller than what we identified. Although our study was conducted during the early decentralization of HIV treatment in Zimbabwe, initial KS diagnosis and treatment in Zimbabwe, and other African countries, remain largely decentralized and our findings remain relevant in the present day.5Training Zimbabwean healthcare providers in KS diagnosis and care increased confidence in their clinical abilities and improved retention in care. Basic knowledge regarding early KS diagnosis, the use of biopsy, treatments, such as chemotherapy, and symptom palliation should be included in standard curricula for primary healthcare providers in Zimbabwe and other African countries with a high burden of AIDS‐KS. Improvements in morbidity and mortality caused by AIDS‐KS in sub‐Saharan Africa will likely require a multi‐pronged approach that includes oncology training of health workers in rural areas, increased public knowledge of AIDS‐KS, and increased access to affordable cancer diagnostics and effective chemotherapies.No competing interests were reported by any authors.All authors made a considerable contribution to the study and manuscript. KRS contributed to data interpretation and manuscript writing and revision. MB and TBC were significantly involved with study design, data acquisition and interpretation, and manuscript writing and revision. CMM, JSK, EAFS and JH contributed substantially to study design, data acquisition and interpretation, and manuscript revision. SM supplied time and effort towards study design, data analysis and interpretation, and manuscript writing and revision. MM contributed significantly to data analysis and interpretation and manuscript revision. MM, FJ, SF, DDC, CM, BM, CB, PG, RM, MM and IG were substantially involved in data acquisition and interpretation, and manuscript revision. All authors have approved the final version of this manuscript to be published and agree to the accountability of all aspects of the work completed.Funding was provided by the National Cancer Institute, National Institutes of Health: grant 1R01CA172050. The funders had no role in study design, data collection or interpretation.The findings of this study are solely the responsibility of the authors and do not necessarily represent the official views of NCI or NIH.File S1: SIKO KS Clinical Management Manual.Click here for additional data file.Information on file format. A PDF file containing the manual used during the described intervention for KS diagnosis and management.Click here for additional data file.


High glycated-hemoglobin (HbA1c) levels correlated with an elevated risk of adverse cardiovascular outcomes despite renin-angiotensin system (RAS) inhibition in type-2 diabetic (T2DM) patients with reduced ejection fraction. Using the routine biopsies of non-T2DM heart transplanted (HTX) in T2DM recipients, we evaluated whether the diabetic milieu modulates glycosylated ACE2 (GlycACE2) levels in cardiomyocytes, known to be affected by non-enzymatic glycosylation, and the relationship with glycemic control.We investigated the possible effects of GlycACE2 on the anti-remodeling pathways of the RAS inhibitors by evaluating the levels of Angiotensin (Ang) 1–9, Ang 1–7, and Mas receptor (MasR), Nuclear-factor of activated T-cells (NFAT), and fibrosis in human hearts.We evaluated 197 first HTX recipients . All patients were treated with angiotensin-converting enzyme inhibitor (ACE-I) or angiotensin receptor blocker (ARB) at hospital discharge. Patients underwent clinical evaluation . Biopsies were used to evaluate ACE2, GlycACE2, Ang 1–9, Ang 1–7, MasR, NAFT, and fibrosis.GlycACE2 was higher in T2DM compared tonon-T2DM cardiomyocytes. Moreover, reduced expressions of Ang 1–9, Ang 1–7, and MasR were observed, suggesting impaired effects of RAS-inhibition in diabetic hearts. Accordingly, biopsies from T2DM recipients showed higher fibrosis than those from non-T2DM recipients. Notably, the expression of GlycACE2 in heart biopsies was strongly dependent on glycemic control, as reflected by the correlation between mean plasma HbA1c, evaluated quarterly during the 12-month follow-up, and GlycACE2 expression.Poor glycemic control, favoring GlycACE2, may attenuate the cardioprotective effects of RAS-inhibition. However, the achievement of tight glycemic control normalizes the anti-remodeling effects of RAS-inhibition.Trial registration: https://clinicaltrials.gov/ NCT03546062.The online version contains supplementary material available at 10.1186/s12933-022-01573-x. Therapy to inhibit the renin-angiotensin system (RAS) is effective and well-tolerated in diabetic and nondiabetic patients with heart failure –4, regarTo date, no evidence has demonstrated the role of hyperglycemia on the expression of GlycACE2 in cardiomyocytes of human beating diabetic hearts, which could impact the binding of Ang-I and Ang-II and, consequently, the expression levels of Ang 1–9 and Ang 1–7 in humans. So far, in biopsies of transplanted hearts , we obtaSince January 2010, we have been conducting a prospective study (NCT03546062)  under ALThe study population was divided into two groups according to whether patients did or did not have T2DM before the transplantation. The study included patients with T2DM for at least 6 months before HTX, without diabetic complications, following ISHLT guidelines . PatientThe internationally accepted evaluations were recorded after HTX at weeks 1, 24, and 48 . At 12-month follow-up, the patients were divided, as post hoc analysis, into non-T2DM, T2DM with good glycemic control (HbA1c < 7%), and T2DM in poor glycemic control (HbA1c ≥ 7%) groups, based on the mean HbA1c evaluated quarterly . Moreove3 from the left ventricular free wall. Tissues were immediately treated and analyzed as described previously   for 16 h. Sections, incubated using Alexa Fluor 488 or 633 secondary antibodies diluted at 1:1000 in blocking solution for 1 h at RT, were then quenched for autofluorescence using the Vector TrueVIEW Autofluorescence Quenching Kit . To ensure that what appears to be specific staining was not caused by non-specific interactions of immunoglobulin molecules with the sample, sections from non-T2DM and T2DM patients were incubated with blocking solution, supplemented with a non-immune immunoglobulin IgG antibody, followed by a secondary antibody incubation for 1 h at RT. All samples were stained with DAPI  for 10 min before mounting in Vectashield Mounting Medium . Using a Zeiss LSM 710 confocal microscope, all slides were imaged with a plan apochromat X63 (NA1.4) oil immersion objective.g for 10 min at 4 °C to collect the supernatant. 50–60 μg of sample proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. Membranes were incubated for 1 h at RT with blocking buffer solution, TBS-T containing 20 mM Tris, pH 7.6, 100 nM NaCl, 0.1% Tween-20, and 5% non-fat dry milk under gentle shaker. Membranes were then incubated with specific primary antibodies against ACE2  or GlycACE2  at 4 °C overnight, followed by incubation with peroxidase-conjugated secondary antibodies for 1 h at RT. In this study two antibodies have been used in order to distinguish GlycACE2 from ACE2. The antibody for ACE2  detects a band size in human tissues at 120–135 kDa, as reported by the manufacturer. The antibody for GlycACE2  detected a band at 120–135 kDa, and was also tested with an aliquot of recombinant human ACE2 (hACE2) (MW = 100 kDa) after in vitro glycation  buffer, 30 mmol/l Tris–HCl, pH 8.8), were added to tissues cut into small pieces. Tissues homogenized with a Precellys 24 system (Bertin Technologies) were centrifuged at 800×lycation . As repolycation . Membran−ΔΔCt method by using GAPDH  as control.Total RNA was isolated from human heart sample homogenates, according to the manufacturer’s protocol, by using RNeasy Mini kit  and was quantified with NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific). Genomic DNA (gDNA) contaminations were removed from heart samples and mRNA was converted to cDNA by using QuantiTect Reverse Transcription kit —Reverse Transcription with Elimination of Genomic DNA for Quantitative, Real-Time PCR Protocol—and Gene AMP PCR System 9700 (Applied Biosystems). cDNA were amplified with the CFX96 Real-time System C1000 Touch Thermal Cycler (BIORAD), according to the protocol “Two-Step RT-PCR (Standard Protocol)”. Particularly, QuantiTect SYBR Green PCR Kit  and QuantiTect Primer Assays were used in order to detect human ACE2  gene expression, quantized with 2MasR and NFAT  ELISA Kit, abx520337, Abbexa) levels in tissue extracts from heart biopsies , according to the manufacturer’s protocol for tissue homogenates. Briefly, tissues were rinsed in ice-cold PBS, cut into small pieces, and homogenized in fresh 2D lysis buffer with a Precellys 24 system (Bertin Technologies). The resulting suspension was centrifuged for 5 min at 10,000×g and the clarified surnatant was incubated in the pre-coated plates with specific anti-Ang 1–7, -Ang 1–9, -MAS1 and -NFAM1 antibodies, following the manufacturer's instruction. For each sample, the Optical Density (OD) is measured spectrophotometrically at 450 nm in a microplate reader (Bio-Rad) and Ang 1–7, Ang 1–9, MAS1 and NFAM1 levels in samples determined by plotting the absorbance values against concentrations of each standard curve. The assessment of Ang 1–7 and Ang 1–9 content was performed by using ELISA kits with high specificity in the detection to avoid significant cross-reactivity or interference between Ang 1–7 or Ang 1–9 and their analogs as reported in the specific datasheet. In detail, during the reaction, human Ang 1–7 or Ang 1–9 in samples compete with a fixed amount of human Ang 1–7 or Ang 1–9 on the solid phase supporter for sites on the biotinylated detection Ab specific to Human Ang 1–7 or Ang 1–9. No significant cross-reactivity or interference between human Ang 1–7 and Ang 1–9 was observed.Enzyme-linked immunosorbent assay (ELISA) colorimetric kits were used for the determination of human Ang 1–7, Ang 1–9, For morphological diagnosis, sections (4 μm thick) were stained with hematoxylin and eosin (H&E). Masson's Tricromica Stain was used for the differential staining of collagen. All stained samples were examined under light and digital microscopes. The content of collagen fibers relative to the total adjacent normal tissue by image analysis using the software Zen 3.3  was also evaluated.Data are expressed as mean ± SD for continuous variables and percentage for categorical variables.Two-way repeated-measures ANOVA was conducted to determine the differences in cardiac GlycACE2, Ang 1–7, Ang 1–9, MasR, and NFAT levels at baseline and after 12 months in diabetic and non-diabetic patients. Interaction effect was assessed to determine within-group changes and between-group differences at baseline and 12 months. The Shapiro–Wilk test was used to assess the normality of the data. A multiple regression model was used to assess changes in GlycACE2 levels by age, sex, BMI, and glycated hemoglobin levels. A P-value < 0.05 was considered statistically significant. Data were analyzed with SPSS software (version 23).Characteristics of the HTX recipients and donors are shown in Table −ΔΔCt = 3.1 ± 0.7 and 3.5 ± 0.6, both P < 0.05 vs nondiabetic) compared to nondiabetic patients (2−ΔΔCt = 1.2 ± 0.3) , as well as diabetic with high glycemic control   and final (44 to 48 weeks) Fig.  evidenceTo evaluate the activity of GlycACE2, we analyzed Ang 1–9, Ang 1–7, MasR, and NAFT in T2DM and non-T2DMventricular specimens Fig. . Ang 1–9). Finally, there were no differences among patients treated with ACE-I and ARB  higher level of GlycACE2 expression in cardiomyocytes of TD2M correlated with glycemic control expressed as HbA1c levels; (2) non-enzymatic glycosylation can affect GlycACE2 levels with concomitant impairment of ACE2 activity in cardiac biopsies from T2DM patients, as evidenced by the reduced levels of Ang 1–9, Ang 1–7, and MasR; (3) the specific pathogenic contribution of GlycACE2 correlated with myocardial fibrosis and development of impaired heart function; and (4) good glycemic control, as a mean HbA1c of < 7% during 12 months after HTX, improved the anti-remodeling effects of the RAS inhibition by reducing myocardial levels of GlycACE2 as well as increasing levels of Ang 1–9, Ang 1–7, and MasR. Moreover, regardless of the pathogenic causes of HF, explanted hearts from T2DM patients had high levels of GlycACE2 and fibrosis and reduced levels of Ang 1–9, Ang 1–7, and MasR .Experimental studies evidenced that Ang 1–7 treatment ameliorated left ventricular remodeling and dysfunction in diabetic rats by attenuating myocardial fibrosis, myocardial hypertrophy, and myocyte apoptosis via the MasR/NAFT pathways , 21. FurFirst, our real-life study was based at only a single institution and had a small number of subjects, so we need to extend our observations to a larger cohort of patients. Second, immunosuppressive therapy per se could affect the molecular mechanisms of RAS inhibition. However, regression analysis showed that progressive decreases of ANG 1–7, ANG 1–9, and MasR in T2DM recipients were independent of the immunosuppressive state, including covariables such as polyclonal anti-lymphocyte antibodies, cyclosporine, tacrolimus, mycophenolate mofetil, everolimus, and prednisone. Moreover, the findings that higher levels of GlycACE2 and reduced ACE2/Ang 1–7, 1–9/MasR axis with a parallel increase of myocardial fibrosis were observed only in diabetic patients, as well as the improvement of anti-remodeling effects of RAS inhibition in diabetic patients with HbA1c levels < 7% suggest a pivotal role of glycemic control in the efficacy of both ACE-I and ARB therapy. Third, our molecular data come from cross-sectional analyses.Finally, in the current study we did not measure the plasma renin activity (PRA) levels after the treatment with ARB or ACE-I. Despite this, the PRA levels and consequently the PRA-guided therapy have been proposed to evaluate the effects of the ACE-I or ARB on the treated patients .Poor glycemic control may favor glycation of ACE2 and reduce the cardiac protection of RAS-inhibition. Furthermore, our study suggests that the achievement of tight glycemic control normalizes there modeling effects of ACE-I and ARB therapy. Finally, our data assume an important role in understanding the effects of RAS-inhibition on the molecular mechanisms of the diabetic cardiomyocyte, in light of recent evidence suggesting that the RAS should be regarded as both a circulating and cellular organized hierarchical angiotensin network linked by characteristic enzymatic reactions , 28.Competency in patient care and procedural outcomes: good glycemic control, as evidenced by HbA1c levels < 7%, improves anti-remodeling effects of RAS-inhibition therapy in diabetic patients.Translational outlook: longer-term follow-up and direct comparisons with other therapy as sacubitril/valsartan will help define the role of anti-remodeling therapy to prevent diabetic cardiomyopathy.Additional file 1: Figure S1. Western blot analysis of GlycACE2. Representative immunoblotting of recombinant hACE2 protein  after in vitro long-term exposure to glucose 120 mM was separated on SDS-PAGE by using 7% gels in reducing and non-reducing conditions and then transferred on nitrocellulose membrane. Membrane was incubated with specific primary antibody against GlycACE2 (1:1000) . Molecular weight indicators are displayed at the center. Figure S2. Ejection fraction, TAPSE, and the E/e′ at week 1  and week 48 (follow-up) in nondiabetic and diabetic patients treated with ACE-inhibitors (ACE-I) and angiotensin receptor blocker (ARB). Data are mean ± SD. *P < 0.05 vs non-diabetics, §P < 0.05 vs basal values. Figure S3. GlycACE2 at week 1  and week 48 (Follow-up) in the diabetic patients with good glycemic control (HbA1c < 7%) and diabetic patients with poor glycemic control (HbA1c ≥ 7%) treated with ACE-inhibitors (ACE-I) and angiotensin receptor blocker (ARB). Data are mean ± SD. *P < 0.05 vs non-diabetics, §P < 0.05 vs basal values. Figure S4.A Angiotensin-1–9 (Ang-1–9), Ang 1–7, Mas receptor (MasR), Nuclear factor of activated T-cells (NFAT), in explanted hearts (HTX) at week 1  and week 48 (Follow-up) from HTX, in nondiabetic and diabetic patients, treated with ACE-inhibitors (ACE-I) and angiotensin receptor blocker (ARB). B Ang-1–9, Ang 1–7, MasR, and NFAT, in explanted hearts (HTX) at week 1  and week 48 (Follow-up) from HTX,in the diabetic patients with good glycemic control (HbA1c < 7%) and diabetic patients with poor glycemic control (HbA1c ≥ 7%) treated with ACE-inhibitors (ACE-I) and angiotensin receptor blocker (ARB). Data are mean ± SD. *P < 0.05 vs non-diabetics, §P < 0.05 vs basal values. Figure S5. Fibrosis percentage in explanted hearts (HTX), at week 1  and week 48 (follow-up) from HTX, in the diabetic patients with good glycemic control (HbA1c < 7%) and diabetic patients with poor glycemic control (HbA1c ≥ 7%) treated with ACE-inhibitors (ACE-I) and angiotensin receptor blocker (ARB). Data are mean ± SD. *P < 0.05 vs non-diabetics, §P < 0.05 vs basal values. Table S1. Multivariate linear regression analysis with GlycACE2 as dependent variable.


Kaposi sarcoma is a vascular tumor highly related to human herpesvirus-8 and Kaposi sarcoma–associated herpesvirus. Kaposi sarcoma usually manifests as skin or mucosal lesions; involvement in visceral organs such as the gastrointestinal tract is rare. Kaposi sarcoma can occur in immunocompromised patients receiving immunosuppressive therapy, in which case it is known as iatrogenic Kaposi sarcoma or drug-induced Kaposi sarcoma. Intestinal Kaposi sarcoma in patients with inflammatory bowel disease is extremely rare.A 46-year-old East Asian male with recently diagnosed Crohn’s disease was administered azathioprine and prednisolone; however, the patient complained of persistent abdominal pain and diarrhea following treatment. Endoscopy revealed small bowel Kaposi sarcoma. The patient was treated with systemic chemotherapy successfully without relapse.This is the fifth case of Kaposi sarcoma developed over the small intestine in a patient with Crohn’s disease following administration of immunomodulators. Additionally, this case indicated that even short-term immunomodulator use can induce Kaposi sarcoma in patients with inflammatory bowel disease. Thus, in patients with inflammatory bowel disease, if symptoms are aggravated or do not abate after immunomodulators prescription, and before intending to upgrade immunomodulators, endoscopy should be considered. Finally, chemotherapy can also be considered if both medication withdrawal and surgical intervention are not feasible. Kaposi sarcoma is an opportunistic, angioproliferative tumor, closely related to human herpesvirus-8 (HHV-8) and Kaposi sarcoma–associated herpesvirus (KSHV). In addition to widely recognized cutaneous or mucosal manifestations, visceral involvement has been described. KS that occurs in immunocompromised patients receiving immunosuppressive therapy, not including KS that is associated with human immunodeficiency virus (HIV), is known as iatrogenic KS or drug-induced KS , 2.Inflammatory bowel disease (IBD) is a condition of chronic inflammation of the gastrointestinal tract. Patients with IBD usually require one or more immunomodulators if they are resistant to basic treatment. Intestinal KS following immunomodulation in patients with IBD –6 is a rHere, we report a case of KS that developed in the small intestine in a patient with Crohn’s disease following short-term administration of immunomodulators.A 46-year-old East Asian male with a history of Crohn’s disease  reported to have no psychosocial disorder nor significant family history. He was treated with immunomodulators including an initial treatment of azathioprine 50 mg and prednisolone 15 mg twice daily, and mesalazine. Owing to remission of the clinical symptoms of Crohn’s disease, prednisolone was gradually tapered off; prednisone use covered a total period of 3 months.Following 4 months of azathioprine and 3 months of prednisolone use, the patient complained of persistent abdominal pain and diarrhea. Endoscopy in March 2019 revealed scarring in the colon and terminal ileum. Furthermore, inflammatory polypoid lesions were noted in the terminal ileum that were not present in a previous examination  enterography and endoscopy were used when symptoms of Crohn’s disease flared up. The patient did not relapse for more than 2 years.Iatrogenic KS is a variant of KS that is associated with immunosuppression or transplantation that develops after immunosuppressive therapy. Classic KS presents predominantly with multiple vascular cutaneous and mucosal nodules, whereas iatrogenic KS is more likely to involve the lymph nodes, mucosa, and visceral organs, including of those of the gastrointestinal tract, and sometimes absence of manifestation in the skin , 7. KS-aGiven that intestinal KS is located in the submucosa with intraluminal growths, endoscopy typically reveals an intraluminal nodular or polypoid tumor with a reddish, violaceous brown or blue color , 5. PseuThere are no specific staging systems or treatment guidelines for iatrogenic KS . PresentIntestinal KS in patients with IBD following administration of immunomodulators is extremely rare –5. We seWe found the present case worthy of discussion. Firstly, the patient received a combination of the immunomodulators azathioprine and prednisolone for 3 months and then azathioprine alone for 1 month. The period of exposure to the immunomodulators prior to the development of KS was relatively short. By contrast, the average period of exposure to immunomodulators in the cases in the literature of patients with KS and IBD ranged from 1 to 2 years. A few cases of patients with ulcerative colitis reported administration of immunomodulators for just 1–3 months prior to the development of intestinal KS . We beliIn summary, we reported a case of isolated intestinal KS, a rare complication of IBD, that manifested as submucosal polypoid lesions that was diagnosed using endoscopy. Histopathology was required for a definite diagnosis and to exclude other diseases. Additionally, this case indicated the possibility that even short-term immunomodulators can induce KS in patients with IBD. Thus, in patients with IBD, if symptoms are aggravated or do not abate following immunomodulator administration and before we intended to upgrade immunomodulators, endoscopy should be considered. Finally, chemotherapy could also be considered if medication withdrawal and surgical intervention are not feasible.


In the past decade, only a small number of papers have elaborated on the application of physiologically based pharmacokinetic (PBPK) modeling across different areas. In this review, an in-depth analysis of the distribution of PBPK modeling in relation to its application in various research topics and model validation was conducted by text mining tools. Orange 3.32.0, an open-source data mining program was used for text mining. PubMed was used for data retrieval, and the collected articles were analyzed by several widgets. A total of 2699 articles related to PBPK modeling met the predefined criteria. The number of publications per year has been rising steadily. Regarding the application areas, the results revealed that 26% of the publications described the use of PBPK modeling in early drug development, risk assessment and toxicity assessment, followed by absorption/formulation modeling (25%), prediction of drug-disease interactions (20%), drug-drug interactions (DDIs) (17%) and pediatric drug development (12%). Furthermore, the analysis showed that only 12% of the publications mentioned model validation, of which 51% referred to literature-based validation and 26% to experimentally validated models. The obtained results present a valuable review of the state-of-the-art regarding PBPK modeling applications in drug discovery and development and related fields. TM and PK-Sim® have changed their course from being commercial to open access platforms [Modeling and simulation (M&S) is a proven scientific approach that has a profound impact on drug discovery and development processes. Physiologically based pharmacokinetic (PBPK) models represent mechanistic models, which integrate a series of different mathematical equations describing the processes a drug undergoes in a human  organism. Since PBPK models are based on known anatomical and physiological data, they are very useful for the prediction of pharmacokinetic (PK) and pharmacokinetic-dynamic (PK/PD) behavior of drugs and/or chemicals. The term PBPK modeling dates back to 1937 when Teorell introduced mathematical equations to describe drug concentrations over time in blood and body tissues ,3. The elatforms . This islatforms ,40. Howelatforms .In the past 10 years, a large number of review articles on PBPK modeling have been published; however, only a small number of papers focus on the application of PBPK modeling across different areas. Some of them cover only a specific therapeutic area ,45,46,47Due to a large number of publications on PBPK modeling and considering the versatility of applications, the traditional literature review on this topic would be quite time-consuming. Computational text mining tools, on the other hand, are advantageous because a large set of textual data can be analyzed in a short period of time. Text mining uses natural language processing (NLP) to transform the unstructured text in documents and databases into normalized, structured data suitable for analysis . Text miThe aim of this review was to use text mining tools to conduct an in-depth analysis of literature sources on PBPK modeling published in the last 10 years in relation to the application fields of these models in different therapeutic areas, target species, routes of administration etc. Furthermore, the analysis addressed the proportion of publications that include model validation and distribution of different modeling software packages across the extracted publications.Text mining of the literature was performed using Orange 3.32.0 software , an openAccording to the selected setup, each article included in the text mining analysis had to meet the following criteria: (1) to be published in English, (2) to have abstract available for analysis, (3) to be published between 2012 and 2022, (4) to contain at least one of the selected keywords either in the title, abstract or keywords, and (5) to present either an original research article, white paper, review article, perspective, book or supplement articles. This last criterion included reviewed pre-prints published online ahead of print. Other types of manuscripts such as tutorials, conference papers, workshops, commentaries, editorials, protocols, pre-prints that precede a formal review process etc. were excluded from further analysis.Several pre-processing steps were applied to transform the textual data into numerical features that can subsequently be used for further analysis. The preprocessing of text helps to split the raw data into smaller tokens (words or units) and understand the data. Four methods were used to preprocess the data: transformation, tokenization, filtration and and-gram ranging. Within the transformation method, the lowercase option was selected, which transforms all words into lowercase. Additionally, URLs were removed from the text. Secondly, with the help of the tokenization method, the text was split into smaller elements . The “regular expression” (Regexp) technique was selected to omit punctuations from the data. The expression pattern was used to single out only those words that have more than three letters. Next, the data were filtered by removing the so-called “stop words” that are commonly used words in a given language but have no particular meaning. For this purpose, a text file containing the list of the most commonly used “stop words” including prepositions, pronouns, conjunctions etc. was created. The list of “stop words” was further supplemented with words that are not relevant to the analysis and often occur within the text such as: “pbpk”, “model”, “based”, “physiologically”, “pharmacokinetic”, “modeling”. Additionally, filtering out the numbers option was selected for filtering out whole numbers from the text. The last input in the filtration method was the selection of the 100  most frequent tokens. Afterwards, the option WordNet Lemmatizer was chosen for stemming and lemmatization to words. Lastly, the default value of one and two grams was kept for the n-gram range.p-value. The p-value was set to 0.05, meaning that words with a p-value lower than 0.05 are significant for a selected subset compared to the entire corpus. The most relevant keywords were identified using the extract keyword widget, and the word importance was calculated with the “term frequency-inverse document frequency” (TF-IDF) method. The TF-IDF method computes the frequency of each term in the document and weights it by the opposite of the term’s frequency in the whole corpus. The terms with the highest scores were submitted to further analysis.The word cloud widget was used to visually display the most frequently mentioned words (tokens) in the text, where the size of the word is proportional to the frequency of their mention in the text. This widget outputs a list of words sorted by their frequency. Statistically significant words for the entire corpus, as well as for each topic, were identified by connecting the bag of words widget with the word enrichment widget and corpus viewer tool. The significance of each word was determined based on the Topic modeling discovers abstract topics in a textual corpus based on a cluster of words and phrases found in each document and their respective frequency. A document typically contains multiple topics in different proportions; thus, the widget also reports on the topic weight per document. In this analysis, the Latent Dirichlet Allocation (LDA) method was chosen for topic modeling. The most important model parameter, which has to be determined in advance, is the number of topics . Based oIn Using the predefined search criteria, the PubMed widget found 4332 publications. Although the PubMed widget was set to extract only those papers published in the past decade, the widget extracted all papers that contained the assigned keywords since 1977. Due to this failure of the widget, the retrieved articles were manually screened. After excluding 1427 papers based on the year of publication, in total 2905 papers were included for further analysis.The selectivity of the PubMed widget towards the search criteria was rather good, since almost all of the extracted papers contained at least one of the set keywords either in the title, abstract or within the article’s keywords. The only exception was five articles that contained the keyword “PBBM”  because this abbreviation is also used for other phrases such as “porous bovine bone mineral” or “phosphate-buffered basal media”. Further, those articles that did not satisfy the pre-set eligibility criteria (available abstract and appropriate manuscripts) were excluded, and in total 2699 papers were included for the final text mining analysis. Regarding the distribution of the selected publications per year , the numWith the text preprocessing widget, 2699 text documents were converted into 74,635 tokens of 91 types. Using the word cloud option, all tokens were visually displayed .The most frequently mentioned words in the 2699 analyzed abstracts are presented in Since the aim of this review was to investigate the versatility of PBPK modeling applications across retrieved publications, by using the extract keyword option and RegExp filter of the corpus viewer, the presence of certain words/phrases was measured. The obtained results a revealeThe analysis was extended to assess the proportion of published studies that include PBPK model validation. For this purpose, only original research articles were analyzed. Manual screening of the collected publications pool  retrieved 1929 original research articles. Incorporation of the words “validate” and “validation” in the RegExp filter of the corpus viewer widget revealed that, out of the 1929 original research articles, the selected words were present in only 232 articles. Such results indicate that only a small proportion of publications included the important step of PBPK model validation. The additional manual search of these 232 articles revealed that most of the published PBPK models were validated based on literature data (51%), and 26% were verified using experimental data. The analysis also revealed that 10% of the published models were validated using both literature and experimental data, while 13% of the extracted articles contained no information on the model validation procedure in the abstracts. Lastly, the original research articles were evaluated in relation to the used platform/software for model development. For this purpose, the selected keywords that represent common PBPK modeling software packages were incorporated into the RegExp filter of the corpus viewer widget. The results indicated that the word “simcyp” was mentioned in 11% of the analyzed articles, followed by the word “gastroplus” in 5% and “pk-sim” in 3% of the articles. Publications related to the use of other software such as MATLAB, Stella, NONMEN, WinNonlin, Berkeley Madonna and Simbiology were less frequent. The obtained results are in line with the review conducted by El-Khateeb et al., which indicated the prevalence of Simcyp platform in 55% of the published papers, followed by GastroPlus and PK-Sim . It shouBy using the so-far described method, it was possible only to determine in how many papers a certain word and/or phrase appeared, without explaining the context in which it was used. Therefore, the topic modeling widget was used to identify specific topics within the retrieved abstracts.The first five topics set by the LDA algorithm were considered as adequate representatives of the whole corpus based on the good values for log perplexity (16.7) and topic coherence (0.44) . The 10 Regarding the probability of occurrence of a certain topic in each publication, manual counting and screening of the retrieved articles revealed that 711 articles (26%) belong to topic 1, followed by topic 5 presented in 671 articles (25%), topic 2 in 496 articles (20%), topic 4 in 445 articles (17%) and topic 3 in 327 articles (12%). These results coincide with the results of the MTP value obtained with the MTP widget option. The growth in the number of publications within each topic during the past 10 years is shown in These results suggest that topic 1 mainly refers to the utilization of PBPK modeling during early drug development, for the optimization and screening of promising compounds with desired/acceptable PK properties. Additionally, the words “extrapolation”, “rat” and “specie” imply that articles within topic 1 use PBPK in conjunction with in vitro- in vivo extrapolation (IVIVE) for the extrapolation of drug PK data across species. Furthermore, topic 1 clusters the articles that mention the PBPK modeling application for the assessment of human health risk associated with exposure to certain drugs/environment chemicals/mixtures of chemicals, as observed from the frequency of the words “health”, “risk” and “toxicity”.In relation to the specific keywords observed within the word cloud for topic 1 , the extNext, the distribution of PBPK models in relation to the prediction of human exposure to various compounds via different pathways was investigated. The results showed that the majority of the analyzed publications used PBPK modeling for the prediction of human exposure to drugs/chemicals following oral ingestion (25%), of which the majority represents ingestion through water (11%) and fewer ingestion through food (9%). Except for the oral route of administration, a large number of articles describe the use of PBPK modeling for the prediction of certain chemicals/pollutants/drugs exposure through inhalation (15%), whereas the intravenous and topical exposures are not frequently addressed. Regarding the application of PBPK modeling for species extrapolation, the majority of articles describe the use of human models, with the word “human” being more present in the extracted publications, followed by “rat” models.As for the employment of PBPK modeling for the prediction of drugs/chemicals PK properties, the majority of articles mention PBPK models in terms of predicting drugs/chemicals blood/plasma concentration (56%), followed by the prediction of tissue concentration (37%) and urine concentration (only 11%). Furthermore, it was observed that the majority of publications within topic 1 describe the use of PBPK models for the prediction of drugs/chemicals excretion/clearance and metabolism (24%), followed by tissue distribution (23%). Since the words “silico” and “vitro” are present in 50% of the articles in topic 1, it can be presumed that the published PBPK models within topic 1 were generated mainly based on in silico and in vitro data as inputs.The steady rise of published papers within topic 1 indicates that PBPK models are valuable tools, and their use in topic 1 related areas has been constantly increasing from 2012 till 2020 . ApplyinAs for topic 2, the results of the word cloud  and wordThus, topic 2 comprises the articles that explain utilization of PBPK modeling in the early clinical development, as a tool to extrapolate or predict PK changes in specific populations, including various disease groups. In the articles within topic 2, PBPK models were mainly used for dose optimization in different populations (26%) and prediction of a drug’s exposure (25%), as seen in The published papers belonging to topic 2 were further divided and analyzed in relation to the distribution of the described PBPK models for different types of diseases . The RegPBPK modeling is especially useful for prediction of drug-disease-related interactions because this approach can incorporate the multitude of pathophysiological changes in organ impairment and their impact on drug bioperformance. In this way, PBPK models can predict the impact of disease on the exposure of drugs that are either renally or hepatically eliminated . With thThe presence  and signSince P-PBPK models are widely used in pediatric drug development, the extracted articles within topic 3 were further analyzed in terms of application of these models in different areas . The resRegarding the distribution of P-PBPK modeling application through different age groups, most of the published P-PBPK were constructed for neonates, followed by toddlers and adolescents . FurtherPBPK modeling alone or in combination with population pharmacokinetics (popPK), has the potential of becoming an integral part of pediatric drug development. Since PBPK models incorporate the physiological and anatomical differences between adults and children, they offer the advantage of predicting a drug’s PK behavior in children and provide more reliable recommendations regarding pediatric dose adjustments when compared to dose calculations based on a child’s age, body weight and surface area. Within the literature, there are several research articles that confirm the benefit of pediatric PBPK models in different areas such as dose selection for children of different age, for optimization of clinical drug trials design, as well as for assessment of potential DDIs ,68,69,70Such results indicate that the articles within topic 4 mostly describe the application of PBPK modeling to predict the potential for drug-drug interactions. The extracted keywords showed that published PBPK models were used to predict both enzyme- and transporter-mediated DDIs, with the word “enzyme” being more present (36%) than “transporter” (27%). Regarding the distribution of PBPK modeling in relation to the prediction of drugs’ ADME properties affected by DDIs, in the majority of published papers, these models were used to predict DDIs at the level of metabolism (30%), followed by absorption (12%), while only 7% of the models dealt with the prediction of DDIs at the level of drug distribution and elimination. By extracting the articles that contained the word “cyp”, and with the help of the extract keywords widget, the most frequent keywords within this subcorpus were analyzed. Further, the utilization of PBPK modeling for the prediction of transporter-mediated DDIs was investigated, by extracting all publications that contained the word “transporter”. The extract keyword widget showed that frequently mentioned words within the subcorpus “transporter” are “intestinal”, “renal”, “liver”, and “clearance” indicating that most of the articles discuss utilization of PBPK modeling for the prediction of DDIs mediated by the transporters in the liver (32%), kidneys (20%) and intestine (17%). Concerning the use of PBPK modeling for the prediction of DDIs in special populations, the prevalence of the words “patient” (25%) and “healthy” (21%) within topic 4 indicates that disease-specific PBPK models are even more frequently used to estimate DDIs than the models for healthy representatives. Additionally, our analysis revealed that only 3% of the published PBPK models within topic 4 was used to predict DDIs in the pediatric population.As seen from The word cloud  and wordThese results indicate that the articles within topic 5 describe the utilization of PBPK modeling tools during formulation development. In this context, PBPK modeling is often referred to as physiologically based biopharmaceutics modeling (PBBM) and denotes a link between biopredictive in vitro dissolution testing and mechanistic oral absorption modeling . BecauseAfter extracting the 290 articles that contained the word “absorption”, it was observed that the occurrence of the word “oral” is 58%. This is in line with the fact that most marketed drugs are administered orally, and therefore the main focus has been directed to develop PBPK models able to describe bioperformance of orally administered drugs . RegardiSensitivity analysis (SA) is one of the key features of PBPK modeling, which is used to assess the sensitivity of the output variables  to the changes in the input variables  . TherefoAs already pointed out, PBPK/PBBM modeling has been increasingly utilized within academic and research institutions, the pharmaceutical industry and regulatory agencies . PBPK/PBIn the last step, estimation of the proportion of original research articles that include PBPK model validation per topic was performed. The analysis showed that the highest percentage of articles that include model validation belongs to topic 2 (37%), followed by topic 1 (26%), topic 4 (20%), and lastly topic 3 and topic 5 with only 9% of such articles. The highest percentage of experimentally validated models was included in the publications within topic 1 (44%), followed by topic 5 (32%), topic 2 (19%), topic 4 (15%) and lastly topic 3 (14%). On the other hand, the largest percentage of the models validated based on literature data was included in the publications that belong to topic 4 (60%), followed by topic 2 (58%), topic 3 (54%), topic 5 (45%) and topic 1 (33%). Such results may seem unexpected considering the fact that topic 1 deals with the utilization of PBPK modeling in the early drug discovery phase when only limited data on drug performance are available. However, it should be emphasized that topic 1 also includes publications related to the use of PBPK modeling for toxicological predictions, environment risk assessment and interspecies scaling, and these publications contributed to the large percentage of experimentally validated models within topic 1.By applying several text mining techniques on a large sample of publications (2699), we managed to extract useful information on the application of PBPK modeling tools in recent years. However, we need to highlight certain limitations of the conducted review analysis. As already pointed out, the software’s integrated PubMed widget failed to extract the articles based on the period of publication, and therefore this task was performed manually. Another annotation concerns the topic modeling widget and its inability to ideally separate the publications within the defined topics. Namely, the widget only extracts the data on the percentage probability that a publication belongs to a certain topic, so one publication may belong to different topics, and this may complicate the overall analysis. Furthermore, regarding the analysis on the mention of model validation in the original research articles, as well as on the utilization of different software tools for PBPK modeling, the obtained results may not represent the actual number/proportion of the relevant publications since the text mining analysis was limited to the articles’ abstracts. Namely, the abstracts rarely mentioned the information of interest for this review. Additionally, we need to note that validity of the obtained results depended on appropriate pre-processing of the text and correct choice of stop words, as well as proper interpretation and linking of the results, which is supported by the authors’ domain knowledge on the studied topic.This paper represents a successful application of text mining tools for thorough review, systematization and interpretation of research trends in PBPK modeling applications. The increasing number of PBPK-related publications over the years demonstrates the evident shift towards wider utilization of PBPK modeling tools in various research fields. The topic modeling analysis confirmed the application of PBPK modeling in diverse areas. The larger proportion of papers published in the last 5 years is related to the application of PBPK/PBBM modeling in formulation development. However, the number of publications related to other topics, such as modeling drug bioperformance in specific or diseased populations (including pediatric population), modeling of drug-drug interactions and modeling of drug properties (including risk assessment and toxicokinetic assessment) during early drug development is also constantly increasing. Although several reviews on PBPK modeling have been published in the past years, covering various modeling aspects, they generally indicate that PBPK modeling has been most commonly used to assess DDI and guide clinical DDI studies ,41,44. T


Despite the growth of molecular diagnosis from the era of Hippocrates, the emergence of COVID-19 is still remarkable. The previously used molecular techniques were not rapid enough to screen a vast population at home, in offices, and in hospitals. Additionally, these techniques were only available in advanced clinical laboratories.The pandemic outbreak enhanced the urgency of researchers and research and development companies to invent more rapid, robust, and portable devices and instruments to screen a vast community in a cost-effective and short time. There has been noteworthy progress in molecular diagnosing tools before and after the pandemic. This review focuses on the advancements in molecular diagnostic techniques before and after the emergence of COVID-19 and how the pandemic accelerated the implantation of molecular diagnostic techniques in most clinical laboratories towardbecoming routine tests. Molecular diagnosis deals with identifying the patterns and alterations in DNA and RNA. These genomic and proteomic patterns are used for comprehending and classifying the broad knowledge of prognosis, therapeutic monitoring, and diagnosis for human healthcare. Molecular diagnostic techniques are an amalgam of molecular techniques with human genetics and medical knowledge. It is a broad term comprising variable fields such as medicine, clinical pathology, forensic testing, epigenetics, immunotherapy, molecular oncology, metagenomics, molecular biology, biotechnology, immunosuppression, toxicology, precision medicine, etc. .In the early 1980s, before the era of molecular diagnosis, clinical laboratories used human disease histories to cure health problems.The timelines of molecular diagnosis techniques are an incredible journey starting from the speculation of any transferrable material to next-generation sequencing technologies. The journey of molecular diagnosis has been reported back to the millennia of Hippocrates, who was credited as being the first person to speculate about the presence of any genetic material to be involved in the transfer. Fast-forward to 1866, the laws of Gregor Mendel demonstrated the inheritance patterns in pea plants . The disIn the early 1990s, Oswald Avery’s transforming principle and Hershey–Chase experiments laid the foundation for comprehending the genetic transformation mechanism. The year 1953 is marked by a breakthrough in demonstrating the structure of DNA by Watson and Crick’s DNA model. With the isolation of DNA polymerase in 1958, the possibility of copying the genetic material was revealed. Karyotyping was first used to identify Down syndrome and trisomy 21, in 1959, and laid the foundation for analyzing the chromosomal aberrations.Taq Polymerase, extending the complimentary short sequences of primer under cyclic conditions [The extraction of Taq Polymerase enabled Frederick Sanger to invent the Sanger sequencing method, in 1980, considered the first generation sequencing approach. Sanger sequencing works by incorporating random chain-terminating dideoxynucleotides during the process of DNA replication . FinallyBRCA1 and BRCA2 in breast cancer.In the late 1990s, the development of kits to test hepatitis C, cytomegalovirus, BK virus, herpes simplex virus (HSV), and Epstein–Barr virus were innovated. The late 1990s have been marked by many advances in molecular diagnosis by clinical aspects such as amplificationand detection of nucleic acid simultaneously, the development of whole genome shotgun sequencing for targeting the bacterial influenza genome, and fluorescence in situ hybridization (FISH), which works on the principle of detecting the required sequence by hybridizing them with specifically designed fluorescent probes and, in 1999,detected the signal under thefluorescent microscope for detection of lungs cancer [Following the advent of PCR, Applied Biosystems invented the first ground-breaking automatic sequencing machine AB370 with the capillary electrophoresis method to increase the proficiency of sequencing . The nexs cancer .E.coli genome was sequenced, in 2005, with 99.9% accuracy using Polony sequencing, which is a multiplex sequencing technique performed on paired-end tags library as a template for emulsion PCR on microbeads to produce the polonies or polymerase colonies [In the early 2000s, most works were already performed by providing the catalyst for an incoming explosive era of modern technology.The turn of the millennium saw massive advancements in information technology in the form of bioinformatics. Then came the era of ground-breaking and remarkable innovation of next-generation sequencing (NGS)with the arrival of NGS, the Genome Sequencer 20 (GS20), which was introduced in 2005–2007 by 454 Life Sciences corporation and became the first sequencer worldwide, later onworking on the process named 454 pyrosequencing . Similarcolonies . Similarcolonies .The yearAfter the advent of NGS and Bioinformatics, molecular diagnostics in clinical labs started to grow. The simple and most common molecular diagnostic techniques used in the clinical laboratory were FISH, PCR, microarrays, MALDI-TOF, ELISA, and nucleotide sequencing .FISH works on the principle of detecting the required sequence by hybridizing them with specifically designed fluorescent probes and detecting the signal under a fluorescent microscope. FISH is used to diagnose specific features in nucleic acids found in tumors, cancers, amniotic fluids, etc. PCR is used to rapidly amplify any target DNA or RNA of humans, virus, or bacteria, in determining pathogenic and non-pathogenic bacteria and in SNPs analysis. It is the basics of diagnostics in the clinical laboratory. Microarrays detect a large mass of genetic material with a high-throughput screening approach which involves multiplex assays mounted in silicon or glass substrates. The microarrays are used for a wide range of medical applications such as detecting chromosomal abnormalities, SNP detection, determination of post-translational modifications, copy number analysis, gene expression, mutation analysis, and finding causative agents of diseases. Many gene ChiP companies are developing microarrays, such as Illumina, Array IT, Agilent, Affymetrix,Applied Microarrays, etc . Matrix-With the emergence of COVID-19, the era of molecular diagnosis has undergone many improvements. Conventionally, CT scan, hematological assays, and RT-PCR were used. However, due to rapidly increasing cases of COVID-19 and the urgent requirement for rapid and precise testing, there was a dire need to come up with some inventions. For instance, CT scans cannot differentiate the type of virus and disease detection in asymptomatic patients. Additionally, this was costly and unavailable at all hospitals . SimilarLoop-mediated isothermal amplification (LAMP) is an alternative to the cumbersome RT-PCR technique. It uses a single temperature, i.e., 60–65 °C, for amplification, which makes the technique much easier compared to RT-PCR and eliminates the need for a thermocycler, thus making it cheaper than RT-PCR. It can amplify the DNA in 25–35 min using polymerase with high DNA-strand replacement activity using 4–8 specific primers . The higBiosensors are the advanced and robust, cost-effective, portable, and simple technology designed for the detection of various biomolecules such as pathogens, proteins, glucose, etc . Several2 copies and LOD of 1.6 × 101 pfu/mL [Many research studies have developed novel biosensors for detecting SARS-CoV-2, such as field-effect transistors (FET). FET is a biosensor developed for SARS-CoV-2, the surface of which is graphene coated. It is then conjugated with the SARS-CoV-2 anti-spike antibody through a probe linker. FET can detect approximately 1–100 fg/mL of the spike protein of SARS-CoV-2 with 2.42 × 101 pfu/mL . Another1 pfu/mL .E genes, ORF1ab COVID, and RdRp-COVID were detected via application ofLSPR and a plasmonic biosensor using photothermal effect. In this process, the converted plasmonic photothermal energy is used to provide stable heat energy for increasing hybridization of RdRp of COVID-19 with the target complementary DNA sequence. The slope graph obtained from the LSPR with photothermal effect was observed to be higher than the LSPR without the photothermal effect, and sensors without PPT showed a false-positive outcome. This sensor has a LOD of 0.2 pM and can differentiate between S-CoV-2 and S-CoV with a high precision rate of 96% [LSPR is a technique for producing optical phenomena when light waves are confined in gold nanoparticles. A coherent localized plasmon oscillation is produced when incident light and the surface electrons in the conduction band interact with each other . Variouse of 96% .This technique detects the SARS-CoV-2 S1 antigen through the SARS-CoV-2 Spike S1 antibody. By using electro-insertion technique, a membrane-engineered kidney cell was modified with the S1 antigen of SARS-CoV-2. A change in potential when the antibody interacts with the required antigen is detected. This biosensor is formulated on eight gold screen-printed electrodes. These printed electrodes are shielded by a polydimethylsiloxane (PDMS) layer with approximately eight wells. A potentiometric device is used to measure the signal after the suspension of membrane and protein solution is added to the wells. The detection limit of this device is 1 fg/mL .−18 M [Another single-step detection field-deployable biosensor using saliva samples was developed based on plasmonic fiberoptic absorbance with LOD of 10−18 M . Similar−18 M .Clustered regularly interspaced short palindromic repeats (CRISPR) is the most significant innovation of the era. It has induced many advances in molecular diagnostics. Previously famous for its use in gene editing, CRISPR has revolutionized the field of diagnostics after the COVID-19 outbreak. CRISPR-Cas is a part of the natural immune system of microbes for protection against foreign material by recognizing them and eliminating them via CRISPR-associated endonuclease Cas enzymes . The twoIn the SARS-CoV-2 DETECTR experiment, the extracted RNA from the test samples is subjected to RT-PCR to increasethe copy number of RNase P, E, and N genes. CRISPR-Cas12 detects the copies of genome sequences and illuminates with a fluorescence signal after cleavage of the reporter dye. This novel detection kit combines RT-LAMP, CRISPR-Cas, and Lateral Flow Assay (LFA) in one process. This CRISPR-based biosensor is developed by Mammoth Biosciences Inc. and Abbott Viral Diagnostics . This teLeptotrichiawadei [The SHERLOCK system is based on the principle of the CRISPR-Cas VI system. SHERLOCK uses the Cas13 endonuclease activity from hiawadei . Recombihiawadei . If the hiawadei . Since thiawadei . Figure Aptamers are artificially made small oligonucleotide or peptide sequences that target specific DNA or RNA of interest. Aptamer-based detection is an emerging technique to target viral infections ,67. SyntMIT is a diagnostic technique that works in the same way as the “Lock and Key Model” of enzyme and substrate reaction and binds to the most predictable structure with high specificity and affinity. MIPs are synthetically designed receptors for binding the complimentary target molecular of specific shape and orientation on a polymer . The creMIPs have become widely employed in recent years for detection reasons, particularly for viral contamination. Ref. , recentlMicroarrays are multifunctional tools that are used in retrospective research on SARS-CoV-2. This tool can study antigen–antibody interactions, pathogenic behavior, cross reactivity between specific species and target proteins, and immunogenic responses to diseases ,77,78. IWith the emergence of COVID-19 many advances in new techniques have been made as explained above. Similarly, many new kits have been approved by FDA for detection of SARS-CoV-19. The purpose of these kits is to provide rapid and robust testing with minimal time. In August 2021, a list of FDA approved many categories of kits varying on the principle such as RT-PCR kit, antigen detection kit, and antibody detection kit for detection of COVID-19 virus as shown in In the history of molecular diagnosis, the SARS-CoV-2 outbreak significantly impacted the transformation and development of molecular diagnostic techniques. This pandemic enhanced the speed of research for survival. Many new technologies have been made, and more inventions are in the development pipeline. According to FDA reports of April 2022, 294 advancements in molecular tests, 90 in antigen depending tests, and 34 serological tests have been approved .Point-of-care (POC) tests are the medical testing performed near the point of care where the point of care is the patient. POC are portable, cost-effective devices, with reduced sample processing eliminating the need to transport samples to the laboratory. POC does not require any trained professional to collect samples and can measure symptomatic and asymptomatic patients. Protecting the community from spreading any viral infection can be controlled only when detected early. POC offers the rapid detection of viral presence or host antibody response without laboratory settings needed.It can perform thousands of public tests in a single day. POC analysis can be performed at home, in offices, in mobile vans, in healthcare centers, and in emergency rooms . For SARThe Isothermal amplification process is gaining much attention because it does not require the cycle at different temperatures and viral purification, thus making kits more rapid and easy to use. Many RT-PCR- and RT-LAMP-based POC devices have been developed since the emergence of COVID-19. Abbott Diagnostics Scarborough,Inc., developed the first RT-LAMP-based kit approved by EUA authorization, named the ID NOW COVID-19 test . SimilarIn antigen detection tests, specific antibodies against SARS-CoV-19 structural proteins are used. The antigen approach is faster and more rapid than PCR tests but has the drawback of being less sensitive because there is no amplification . Thus, aAntibodies produced bythe immune system in response to SARS-CoV-19 proteins can be detectedby using developed devices. They detect IgM and IgG antibodies from blood, serum, and plasma within 10–20 min. The list of some EUA-approved POC devices working on the antibody detection principle is shown in Starting from the history to present, molecular diagnosis has revolutionized to a great extent with high-throughput technological advancements. It has become very convenient to study the entire genome with high accuracy and decreasing cost. Presently, many kits and tests are being developed which provide rapid detection as compared to the past, making molecular diagnosis indispensable in the clinical laboratory. As technology is overgrowing rapidly with time, the future of molecular diagnostics seems to be very promising. The wave of the corona accelerated and modernized the field of molecular diagnosis miraculously and analyzing the rapid growth in the pandemic assures us of a bright future ahead. The POC devices and biosensors can be potent candidates for molecular diagnosis because of their robustness. As the pandemics are likely to reoccur, investing efforts in molecular diagnosis seems highly imperative. As a result, the molecular diagnosis will be a central part of medical practice in the upcoming era. Whole genome sequencing will be the gold standard for identifying DNA variations associated with genetic diseases. Nevertheless, there is still a lot to discover before applying these techniques, such as cost-effectiveness, availability of resources, management of equipment, personnel training, and reproducibility. Along with proteomic-based testing, these developments will make further improvements in molecular diagnostics for implementation in hospitals, clinics, and healthcare departments, in public or private sectors.The growth in molecular diagnostic labs before and after the emergence of coronavirus is notable. After the pandemic outbreak, the urge to develop fast and robust techniques exponentially increased. Many techniques have been modified to detect a massive community of coronavirus at home and offices. COVID-19 has boosted R&D struggles to launch innovative, more robust, rapid, precise and economical testing approaches than previously used tools. Currently, RT-LAMP, microarray-based detection, aptamer-based diagnosis, SHERLOCK, SHERLOCKv2, FET Biosensors, cell-based potentiometric diagnosis, molecular imprinting technology, etc. are used for diagnosis. Point-of-care (POC) diagnosis has rapidly increased to screen populations outside the hospitals. These POC devices are robust, portable, and can detect thousands of samples simultaneously. In previous times, the viral epidemic of SARS-CoV and MERS-CoV have led to the advancement in rapid diagnostics. For instance, the genome sequence identification of SARS-CoV-2 lead us toward developing nucleic acid-based diagnostic approaches, which assisted in controlling the outburst of COVID-19. The antibody-based and antigen-based diagnostic approaches helped in further comprehension. There is a lack of detailed research in the development of peptide- and aptamer-based biosensors, which could provide us more sensitivity and specificity in COVID-19 diagnosis, but the era of improvement has started, and yet, there is much to come. According to FDA reports, advancements in molecular tests, antigen-depending tests, and serological tests have been approved since the pandemic outbreak. Thus, the emergence has transformed the scientific community much more than any other life event. The scientific community’s collaboration to overcome the pandemic and break the chain of losing lives has increased the significance of molecular tools and molecular diagnosis.


Beckwith–Wiedemann syndrome  is an inborn overgrowth disorder caused by molecular alterations in chromosome 11p15.5. These molecular changes affect so-called imprinted genes, i.e., genes which underlie a complex regulation which is linked to the parental origin of the gene copy. Thus, either the maternal gene copy is expressed or the paternal, but this balanced regulation is prone to disturbances. In fact, different types of molecular variants have been identified in BWS, resulting in a variable phenotype; thus, it was consented that the syndromic entity was extended to the Beckwith–Wiedemann spectrum (BWSp). Some molecular subgroups of BWSp are associated with an increased embryonic tumor risk and have different likelihoods for specific tumors. Therefore, the precise determination of the molecular subgroup is needed for precise monitoring and treatment, but the molecular diagnostic procedure has several limitations and challenges which have to be considered.Beckwith–Wiedemann syndrome  is a congenital imprinting condition with a heterogenous clinical presentation of overgrowth and an increased childhood cancer risk . Due to the varying clinical presentation encompassing classical, clinical BWS without a molecular diagnosis and BWS-related phenotypes with an 11p15.5 molecular anomaly, the syndromic entity was extended to the Beckwith–Wiedemann spectrum (BWSp). The tumor risk of up to 30% depends on the molecular subtype of BWSp with causative genetic or epigenetic alterations in the chromosomal region 11p15.5. The molecular diagnosis of BWSp can be challenging for several reasons, including the range of causative molecular mechanisms which are frequently mosaic. The molecular basis of tumor formation appears to relate to stalled cellular differentiation in certain organs that predisposes persisting embryonic cells to accumulate additional molecular defects, which then results in a range of embryonal tumors. The molecular subtype of BWSp not only influences the overall risk of neoplasia, but also the likelihood of specific embryonal tumors. Beckwith–Wiedemann syndrome (BWS) is caused by defects in a group of imprinted genes in 11p15.5. Different types of genetic/epigenetic alterations can affect this fine-tuned expression, and result either in an overexpression  or a silencing of imprinted genes. Due to the varying clinical findings of patients with 11p15.5 disturbances, the syndromic entity was extended to the Beckwith–Wiedemann spectrum (BWSp). Several clinical features of BWS overlap with those of other imprinting disorders , Kagami–Ogata syndrome (OMIM 608149), pseudohypoparathyroidism type 1B (OMIM 603233)), and this ambiguity is reflected by molecularly overlapping alterations. Molecular alterations in BWSp patients affect one or both imprinted regions in 11p15.5, which comprise the imprinting control regions 1 and 2 . Molecular changes in the same region also occur in patients with Silver–Russell syndrome (OMIM 180860), but in this growth retardation disorder the molecular alterations are opposite to those that occur in BWSp.Clinically, the term BWSp describes a complex heterogenous and multisystem disease spectrum. It can be diagnosed by clinical assessment and/or molecular validation. The clinical features comprise characteristic developmental anomalies such as midline abdominal defects , enlarged tongue (macroglossia) and overgrowth (gigantism), lateralized overgrowth (hemihypertrophy) and neonatal hypoglycemia (for review: ). Up to 8% of all BWSp patients develop an embryonal tumor during their early childhood but the exact tumor risk depends, however, on the precise causative genetic/epigenetic alteration. Thus, depending on the molecular subtype of BWSp, the childhood cancer risk ranges from 1 to 30%. The main tumor types associated with BWSp are Wilms tumor (nephroblastoma), hepatoblastoma and neuroblastoma . The chromosomal region associated with BWSp was initially identified by rare cytogenetic anomalies in BWSp patients involving the distal short arm of chromosome 11 11p15) ,4. The c ,4. The IGF2  and the long non-coding RNA (lncRNA) H19 , whereas the centromeric imprinted cluster includes the negative cell cycle regulator gene CDKN1C  and the lncRNA KCNQ1OT1 . The genomic imprinting in these clusters is regulated by parental-specific methylation of two domain-specific differentially methylated regions (DMRs) that mark the respective imprinting control regions (IC1 and IC2).Positional cloning efforts led to the identification of several genes in 11p15.5 that were shown to be imprinted and expressed only from the maternally or paternally derived allele. These genes can be grouped into two functionally independent genomic domains . The telIGF2 and H19. It consists of several repetitively arranged binding sites for the zinc finger protein CTCF (OMIM 604167) and Oct4/Sox2 (OMIM 164177/OMIM 184429). On the paternal allele, methylation of the IC1 differentially methylated region (DMR) prevents CTCF binding and, consequently, leads to expressional silencing of H19 and expression of IGF2 on this allele (via specific chromatin looping). The maternal IC1 is unmethylated, thus CTCF binding results in different chromatin looping and H19 is expressed, whereas IGF2 is transcriptionally repressed.The IC1 lies within the telomeric imprinting cluster and is controlled by the H19/IGF2:IG DMR between KCNQ1OT1, an antisense gene of the ion channel KCNQ1 (OMIM 607542). Methylation on the maternal allele inhibits KCNQ1OT1 expression and enables transcriptional access to KCNQ1 and CDKN1C.The centromeric IC2 is controlled by the KCNQ1OT1:TSS DMR. It is located at the transcriptional start site of the lncRNA In contrast to SRS as its molecular counterpart, BWSp is exclusively linked to molecular alterations in 11p15.5. In BWSp patients, all four types of molecular alterations characteristic for imprinting disorders can be identified . These mA molecular defect affecting imprinted genes in chromosome region 11p15.5 can be demonstrated in approximately 80% of clinically characterized patients with a clinical diagnosis of BWSp . HoweverCDKN1C and presumably KCNQ1, as well as transcriptional upregulation of KCNQ1OT1 ).KCNQ1 mutations with an impact on IC2 methylation [NLRP2 OMIM 609364, NLRP5 OMIM 609658 and PADI6 OMIM 610363), which affect components of the oocyte subcortical maternal complex (SCMC). SCMC proteins orchestrate the proper maturation of the oocyte and the early embryogenesis prior to the activation of the fetal genome. Among other functions, some of these proteins are responsible for the maintenance of the imprinting pattern in the early embryo. Accordingly, biallelic maternal variants in these genes are associated with multilocus fetal imprinting defects  is not well understood in the majority of cases. However, environmental factors such as assisted reproduction techniques have been suggested to predispose to aberrant imprinting and the role of genetic causes in some cases is becoming increasingly recognized (for review: ). These hylation ,23. ZFP5hylation ). Howeve review: ).CDKN1C pathogenic variants [BWSp is clinically heterogenous and patients do not necessarily present with all associated features of BWS, nor with a molecular diagnosis . The carvariants . A diagnvariants ,28,29. Tvariants ,31 (TablCDKN1C mutations are associated with tumor development in approximately 7% (neuroblastoma > nephroblastoma) [The cumulative risk of BWSp patients developing a nephroblastoma is ~5%, but other embryonal tumors including hepatoblastoma, neuroblastoma, adrenal carcinoma and rhabdomyosarcoma occur at lower frequencies. The individual tumor risk depends on the molecular cause of the syndrome. The GOM at the H19/IGF2:IG DMR (IC1) is associated with childhood cancer in 28% of cases (nephroblastoma 24%) and a upd(11)pat has a cumulative childhood tumor risk of approximately 16% (nephroblastoma > hepatoblastoma > neuroblastoma), whereas with a KCNQ1OT1:TSS DMR hypomethylation (IC2 LOM) up to 2.5% of patients develop a tumor (0.7% hepatoblastoma). lastoma) . Patientlastoma) . In the lastoma) ,35. The BWSp-associated cancers are childhood tumors and are thought to arise during early development. These tumor types are caused by rare mutations that arrest the maturation of cell types (in cases of nephroblastomas in approx. 40 genes) in specific cell populations during restricted developmental windows. In contrast to these tumors, adult cancers arise primarily as a consequence of the accumulation of mutations by transforming mature cells into a cancer cell .Nephroblastomas form the major part of BWSp-associated tumors and arise in the kidney directly from the nephrogenic blastema or from embryonic cells that abnormally persist beyond 36 weeks of gestation (nephrogenic rests) with a preferentially perilobar rather than intralobar histology. During normal development of the kidney, renal vesicles are formed after a mesenchymal to epithelial transformation of the metanephric mesenchyme. These vesicles then expand and differentiate in most of the cell types of the final kidney . NephrobIGF2 and H19 either by GOM at the IC1 or upd(11)pat, have a high frequency (17.3%) of bilateral tumors [IGF2 is causative for the nephrogenic rests. Consequently, the BWSp tumors have additional mutations, e.g., in the transcription factor WT1 (OMIM 194070), in the Wnt pathway regulators beta-catenin  and AMER1  or in the apoptosis master regulator TP53 (OMIM 191170). The mutational spectrum also includes genes involved in miRNA biogenesis  or in chromatin silencing . The specific combination of adverse effects of LOI and gene mutation is partly reflected in the histological subtype of the resulting nephroblastoma pat situation and/or an unfavorable SNP combination might then promote tumor development in IGF2 overexpressing persisting embryonic liver cells.The second most common tumor entity associated with BWSp is hepatoblastoma, which is mainly observed in upd(11)pat cases. In addition to the suggestive iewed in ). The loCDKN1C loss-of-function mutations. Neuroblastomas are neuroendocrine tumors that originate from the developing sympathetic nervous system, resulting in tumor development localized most frequently in the adrenal glands and sympathetic ganglia. The most common causative genomic alteration observed here is a MYCN gene amplification, which is present in approximately 18% of all cases, that can transcriptionally upregulate the histone-lysine N-methyltransferase enzyme Enhancer of zeste homolog 2 , promoting an undifferentiated neuroblastoma tumor phenotype associated with poor clinical outcomes  and cyclin G2  were upregulated alongside the cyclin-dependent kinase inhibitor CDKN1C [MYCN expression results in low CDKN1C expression. BWSp neuroblastomas either have an overexpression of IGF2 (IC1 GOM or upd(11)pat) and/or CDKN1C reduction (upd(11)pat or CDKN1C loss-of-function mutations), which might be the relevant “first hit” for parts of the sympathetic nervous system to pave the road for neuroblastoma development due to a second alteration such as MYCN amplification.Neuroblastomas are the third major BWSp-associated tumor type, occurring in BWSp with IC1 GOM and upd(11)pat, but also in the subgroup with utcomes  associated with BWSp can also be found in a number of tumors in patients without BWS. In non-syndromic nephroblastoma, constitutional 11p15.5 CNV resulting in H19/IGF2:IG DMR hypermethylation could be detected . In thesPLAGL1/ZAC1 (OMIM 603044), MEG3 (OMIM 605636), NDN (OMIM 602117), CDKN1C, IGF2, and H19, whereas KCNQ1OT1 was significantly overexpressed [H19 has been shown to influence the transcription of at least five other genes of the IGN (including Igf2 and Peg1 OMIM 601029) by recruiting the methyl-CpG-binding domain protein 1  that transcriptionally downregulates target genes by bringing repressive histone marks to the differentially methylated regions of these targets [CDKN1C, KCNQ1OT1 overexpression, IGF2 overexpression and H19 reduction. In mammalians CDKN1C has a conserved, likely antagonistic function to IGF2 in the placenta [A LOI at 11p15.5 (IGF2:TSS LOM) was detected in breast (33%) and colorectal (80%) tumor tissues . Since txpressed . The expxpressed . The der targets . This coplacenta . This fuCDKN1C sequencing can be conducted.The molecular heterogeneity of BWSp requires a comprehensive molecular testing approach. By considering the frequencies of the molecular subtypes of 11p15.5 variants, methylation tests addressing specific CpGs in the H19/IGF2:IG DMR and the KCNQ1OT1:TSS DMR have been recommended as first step analyses [However, the detection of imprinting defects and upd(11)pat can be hampered by mosaicism; in particular, upd(11)pat might escape detection because of low-level mosaicism . Thus, The initial treatment, histological classifications and staging criteria of nephroblastoma differs, depending on the geographical location of the patient. Two major treatment strategies have been established, one from the International Society of Pediatric Oncology (SIOP) and the second from the Children’s Oncology Group (COG).The SIOP classification of nephroblastomas consists of three major risk groups ranging from low-risk tumors (completely necrotic), intermediate-risk tumors  and high-risk tumors . According to the SIOP protocol, infants younger than 6 months of age that suffer from a unilateral nephroblastoma obtain an initial—preferentially partial—nephrectomy. All patients with a suspected nephroblastoma older than 6 months of age receive either 4 weeks of preoperative chemotherapy with actinomycin D and vincristine  or 6 weeks of actinomycin D, vincristine and doxorubicin . A percutaneous core needle biopsy is suggested in children 7 years of age and older or children with an uncertain clinical picture to avoid a misdiagnosis of nephroblastoma. After the neoadjuvant chemotherapy, a radical nephrectomy is performed in most BWSp patients with unilateral WT if the preoperative chemotherapy does not support a nephron-sparing surgery (NSS), which is also applied in case of bilateral nephroblastomatosis or WTs .The COG classification of WTs distinguishes anaplastic  and non-anaplastic (favorable histology) WTs. The COG strategy favors a primary  nephrectomy for unilateral renal masses in patients without WT predisposition, but in BWSp a neoadjuvant chemotherapy (without biopsy) with the aim of preserving renal units and subsequent surgery at 6–12 weeks after initiation of chemotherapy is suggested .After surgery, according to SIOP and COG, a risk-based adjuvant chemotherapy and radiotherapy is administered with the help of multiple prognostic factors .Hepatoblastoma (HB) arise from a hepatocyte precursor cell and consists of either epithelial  or epithelial and mesenchymal elements (mixed HB). In addition, 80–90% of HB are observed in children between 6 months and 5 years with a median age of 18 months . TreatmeNeuroblastoma most likely originates from sympathoadrenal progenitor cells within the neural crest that normally differentiate to sympathetic ganglion cells and adrenal catecholamine-secreting cells . MYCN amCurrently, neuroblastoma occurring in BWSp patients are treated like isolated neuroblastoma. Treatment depends on a stratification system that aims to group the patients in different risk groups according to tumor-free survival after treatment. To establish a consensus approach for pretreatment risk stratification, an International Neuroblastoma Risk Group (INRG) task force representing the major pediatric cooperative groups around the world was formed in 2005 . The risTo date, no large series of adults with BWSp have been examined for health issues in later life. The consensus group agreed that BWSp molecular subgroups and patients with classical BWS and no detectable molecular anomaly should be offered an abdominal ultrasound (USS) every 3 months until the age of 7 years, and only BWSp patients with IC2 LOM should not be offered routine USS . This coThe clinical heterogeneity of BWSp and the wide underlying spectrum of molecular alterations present a challenge for clinical and molecular diagnosis. Nevertheless, the precise determination of the molecular subgroup is required as the basis for personalized monitoring and treatment, especially in regard to individual tumor risk. Tumors occurring in BWSp patients do not exactly resemble the corresponding tumor types in non-syndromic patients. They have different combinations of causative (epi)genetic alterations, sometimes reflected in the histology of the resulting neoplasia. Thus, an improved molecular diagnosis of BWSp might lead to further optimized (tumor-)treatment protocols based on the understanding of personalized tumor-driving pathways.