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Rationale & Objective: It is unclear whether the effect of canagliflozin on adverse kidney and cardiovascular events in those with diabetic kidney disease varies by age and sex. We assessed the effects of canagliflozin among age group categories and between sexes in the Canagliflozin and Renal Endpoints in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE) study.Study Design: Secondary analysis of a randomized controlled trial.Setting & Participants: Participants in the CREDENCE trial.Intervention: Participants were randomly assigned to receive canagliflozin 100 mg/d or placebo.Outcomes: Primary composite outcome of kidney failure, doubling of serum creatinine concentration, or death due to kidney or cardiovascular disease. Prespecified secondary and safety outcomes were also analyzed. Outcomes were evaluated by age at baseline (Results: The mean age of the cohort was 63.0 ± 9.2 years, and 34% were female. Older age and female sex were independently associated with a lower risk of the composite of adverse kidney outcomes. There was no evidence that the effect of canagliflozin on the primary outcome (a composite of kidney failure, a doubling of serum creatinine concentration, or death from kidney or cardiovascular causes) differed between age groups (HRs, 0.67 [95% CI, 0.52-0.87], 0.63 [0.48-0.82], and 0.89 [0.61-1.29] for ages Limitations: This was a post hoc analysis with multiple comparisons.Conclusions: Canagliflozin consistently reduced the relative risk of kidney events in people with diabetic kidney disease in both sexes and across age subgroups. As a result of greater background risk, the absolute reduction in adverse kidney outcomes was greater in younger participants.Funding: This post hoc analysis of the CREDENCE trial was not funded. The CREDENCE study was sponsored by Janssen Research and Development and was conducted collaboratively by the sponsor, an academic-led steering committee, and an academic research organization, George Clinical.Trial Registration: The original CREDENCE trial was registered at ClinicalTrials.gov with study number NCT02065791.

Cholesteryl ester transfer protein (CETP) is known to influence HDL-C levels, potentially altering the profile of HDL subfractions and consequently cardiovascular risk (CVR). This study aimed to investigate the effect of five single-nucleotide polymorphisms (SNPs; rs1532624, rs5882, rs708272, rs7499892, and rs9989419) and their haplotypes (H) in the CETP gene on 10-year CVR estimated by the Systematic Coronary Risk Evaluation (SCORE), the Framingham Risk Score for Coronary Heart Disease (FRSCHD) and Cardiovascular Disease (FRSCVD) algorithms. Adjusted linear and logistic regression analyses were used to investigate the association of SNPs and 10 haplotypes (H1–H10) on 368 samples from the Hungarian general and Roma populations. The T allele of rs7499892 showed a significant association with increased CVR estimated by FRS. H5, H7, and H8 showed a significant association with increased CVR based on at least one of the algorithms. The impact of H5 was due to its effect on TG and HDL-C levels, while H7 showed a significant association with FRSCHD and H8 with FRSCVD mediated by a mechanism affecting neither TG nor HDL-C levels. Our results suggest that polymorphisms in the CETP gene may have a significant effect on CVR and that this is not mediated exclusively by their effect on TG and HDL-C levels but also by presently unknown mechanisms.

More than 80,000 new cases of bladder cancer are estimated to be diagnosed in 2023. However, the 5-year survival rate for bladder cancer has not changed in decades, highlighting the need for prevention. Numerous cancer-causing mutations are present in the urothelium long before signs of cancer arise. Mutation hotspots in cancer-driving genes were identified in non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) tumor samples. Mutation burden within the hotspot regions was measured in normal urothelium with a low and high risk of cancer. A significant correlation was found between the mutation burden in normal urothelium and bladder cancer tissue within the hotspot regions. A combination of measured hotspot burden and personal risk factors was used to fit machine learning classification models. The efficacy of each model to differentiate between adjacent benign urothelium from bladder cancer patients and normal urothelium from healthy donors was measured. A random forest model using a combination of personal risk factors and mutations within MIBC hotspots yielded the highest AUC of 0.9286 for the prediction of high- vs. low-risk normal urothelium. Currently, there are no effective biomarkers to assess subclinical field disease and early carcinogenic progression in the bladder. Our findings demonstrate novel differences in mutation hotspots in NMIBC and MIBC and provide the first evidence for mutation hotspots to aid in the assessment of cancer risk in the normal urothelium. Early risk assessment and identification of patients at high risk of bladder cancer before the clinical presentation of the disease can pave the way for targeted personalized preventative therapy.

Melanomas account for only approximately 4% of diagnosed skin cancers in the United States but are responsible for the majority of deaths caused by skin cancer. Both genetic factors and ultraviolet (UV) radiation exposure play a role in the development of melanoma. Although melanomas have a strong propensity to metastasize when diagnosed late, melanomas that are diagnosed and treated early pose a low mortality risk. In particular, the identification of patients with increased metastatic risk, who may benefit from early adjuvant therapies, is crucial, especially given the advent of new melanoma treatments. However, the accuracy of classic clinical and histological variables, including the Breslow thickness, presence of ulceration, and lymph node status, might not be sufficient to identify such individuals. Thus, there is a need for the development of additional prognostic melanoma biomarkers that can improve early attempts to stratify melanoma patients and reliably identify high-risk subgroups with the aim of providing effective personalized therapies.In our current work, we discuss and assess emerging primary melanoma tumor biomarkers and prognostic circulating biomarkers.Several promising biomarkers show prognostic value (eg, exosomal MIA (ie, melanoma inhibitory activity), serum S100B, AMLo signatures, and mRNA signatures); however, the scarcity of reliable data precludes the use of these biomarkers in current clinical applications.Further research is needed on several promising biomarkers for melanoma. Large-scale studies are warranted to facilitate the clinical translation of prognostic biomarker applications for melanoma in personalized medicine.

Mutations found in skin are acquired in specific patterns, clustering around mutation-prone genomic locations. The most mutation-prone genomic areas, mutation hotspots, first induce the growth of small cell clones in healthy skin. Mutations accumulate over time, and clones with driver mutations may give rise to skin cancer. Early mutation accumulation is a crucial first step in photocarcinogenesis. Therefore, a sufficient understanding of the process may help predict disease onset and identify avenues for skin cancer prevention. Early epidermal mutation profiles are typically established using high-depth targeted next-generation sequencing. However, there is currently a lack of tools for designing custom panels to capture mutation-enriched genomic regions efficiently. To address this issue, we created a computational algorithm that implements a pseudo-exhaustive approach to identify the best genomic areas to target. We benchmarked the current algorithm in three independent mutation datasets of human epidermal samples. Compared to the sequencing panel designs originally used in these publications, the mutation capture efficacy (number of mutations/base pairs sequenced) of our designed panel improved 9.6–12.1-fold. Mutation burden in the chronically sun-exposed and intermittently sun-exposed normal epidermis was measured within genomic regions identified by hotSPOT based on cutaneous squamous cell carcinoma (cSCC) mutation patterns. We found a significant increase in mutation capture efficacy and mutation burden in cSCC hotspots in chronically sun-exposed vs. intermittently sun-exposed epidermis (p < 0.0001). Our results show that our hotSPOT web application provides a publicly available resource for researchers to design custom panels, enabling efficient detection of somatic mutations in clinically normal tissues and other similar targeted sequencing studies. Moreover, hotSPOT also enables the comparison of mutation burden between normal tissues and cancer.

Lysine-specific histone demethylase 1 (KDM1A/LSD1) regulates multiple cellular functions, including cellular proliferation, differentiation, and DNA repair. KDM1A is overexpressed in squamous cell carcinoma of the skin and inhibition of KDM1A can suppress cutaneous carcinogenesis. Despite the role of KDM1A in skin and DNA repair, the effect of KDM1A inhibition on cellular ultraviolet (UV) response has not been studied.The ability of KDM1A inhibitor bizine to modify cell death after UVA and UVB exposure was tested in normal human keratinocytes and melanocytes, HaCaT, and FaDu cell lines. KDM1A was also downregulated using shRNA and inhibited by phenelzine in HaCaT and FaDu cells to confirm the role of KDM1A in UVA response. In addition, cellular reactive oxygen species (ROS) changes were assessed by a lipid-soluble fluorescent indicator of lipid oxidation, and ROS-related gene regulation using qPCR. During photodynamic therapy (PDT) studies HaCaT and FaDu cells were treated with aminolaevulinic acid (5-ALA) or HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a) sodium and irradiated with 0-8 J/cmKDM1A inhibition sensitized cells to UVA radiation-induced cell death but not to UVB. KDM1A inhibition increased ROS generation as detected by increased lipid peroxidation and the upregulation of ROS-responsive genes. The effectiveness of both ALA and HPPH PDT significantly improved in vitro in HaCaT and FaDu cells after KDM1A inhibition.KDM1A is a regulator of cellular UV response and KDM1A inhibition can improve PDT efficacy.

ImportanceChanges in postsurgical opioid prescribing practices may help reduce chronic opioid use in surgical patients.ObjectiveTo investigate whether postsurgical acute pain across different surgical subspecialties can be managed effectively after hospital discharge with an opioid supply of 3 or fewer days and whether this reduction in prescribed opioids is associated with reduced new, persistent opioid use.Design, Setting, and ParticipantsIn this prospective cohort study with a case-control design, a restrictive opioid prescription protocol (ROPP) specifying an opioid supply of 3 or fewer days after discharge from surgery along with standardized patient education was implemented across all surgical services at a tertiary-care comprehensive cancer center. Participants were all patients who underwent surgery from August 1, 2018, to July 31, 2019.Main Outcomes and MeasuresMain outcomes were the rate of compliance with the ROPP in each surgical service, the mean number of prescription days and refill requests, type of opioid prescribed, and rate of conversion to chronic opioid use determined via a state-run opioid prescription program. Postsurgical complications were also measured.ResultsA total of 4068 patients (mean [SD] age, 61.0 [13.8] years; 2528 women [62.1%]) were included, with 2017 in the pre-ROPP group (August 1, 2018, to January 31, 2019) and 2051 in the post-ROPP group (February 1, 2019, to July 31, 2019). The rate of compliance with the protocol was 95%. After implementation of the ROPP, mean opioid prescription days decreased from a mean (SD) of 3.9 (4.5) days in the pre-ROPP group to 1.9 (3.6) days in the post-ROPP group (P P P = .02). There was no statistically significant difference in surgical complications. The conversion rate to chronic opioid use decreased following ROPP implementation among both opioid-naive patients with cancer (11.3% [143 of 1267] to 4.5% [118 of 2645]; P P = .02).Conclusions and RelevanceIn this cohort study, prescribing an opioid supply of 3 or fewer days to surgical patients after hospital discharge was feasible for most patients, led to a significant decrease in the number of opioids prescribed after surgery, and was associated with a significantly decreased conversion to long-term opioid use without concomitant increases in refill requests or significant compromises in surgical recovery.

Melanoma is among the most devastating human malignancies. Accurate diagnosis and prognosis are essential to offer optimal treatment. Histopathology is the gold standard for establishing melanoma diagnosis and prognostic features. However, discrepancies often exist between pathologists, and analysis is costly and time-consuming. Deep-learning algorithms are deployed to improve melanoma diagnosis and prognostication from histological images of melanoma. In recent years, the development of these machine-learning tools has accelerated, and machine learning is poised to become a clinical tool to aid melanoma histology. Nevertheless, a review of the advances in machine learning in melanoma histology was lacking. We performed a comprehensive literature search to provide a complete overview of the recent advances in machine learning in the assessment of melanoma based on hematoxylin eosin digital pathology images. In our work, we review 37 recent publications, compare the methods and performance of the reviewed studies, and highlight the variety of promising machine-learning applications in melanoma histology.

Cutaneous squamous cell carcinoma (cSCC) is the second most common human malignancy in the United States. Chronic long-term ultraviolet (UV) exposure drives cSCC development. Early clonal mutation (CM) accumulation is the first step in photocarcinogenesis and is the first known manifestation of field cancerization. Understanding CM accumulation and development is expected to improve the prevention of field cancerization and cSCC. Nevertheless, CMs are poorly understood, and mutations in cSCC have yet to be systematically compared to CM in sun-exposed skin. Key steps for studies to detect and compare CMs are to design optimal targeted sequencing panels and compare mutational hotspot areas. The ideal panel should cover genomic regions containing maximum numbers of mutations within a given number of amplicons. Currently there is no computational tool to help optimize target area selection. Therefore, we aimed to create an algorithm to optimize sequencing target area design for capturing high mutation frequency hotspot areas. An R Shiny web application was created to identify an optimal sequencing target panel from an input mutation dataset and compare the distribution of mutational hotspots. The tool optimizes sequencing target areas based on preset amplicon length by identifying the best fitting panel of amplicons to capture mutations efficiently. Besides identifying optimal sequencing target areas, the developed software also efficiently identifies the most highly mutated genomic areas and compares target area overlaps. The developed algorithm was used to compare the mutational hotspots of cSCC and CM in clinically normal-appearing skin or cSCC from previous publications (cBioPortal, Martincorena et al, Hernando et al, Wei et al, Fowler et al). The current method was more efficient than tested previously available alternative methods by increasing target areas capture efficacy by 1.05 - 8.1-fold. Using the developed tool, we found that frequently mutated areas of CMs in normal skin significantly overlap (p < 0.005) with those of cSCC. Mutational hotspots of normal skin with a history of frequent UV exposure had 1.2-fold greater overlap with cSCC than skin with minimal UV exposure, suggesting the frequently UV-exposed skin carries a greater number of cSCC-related mutations. Although we found that normal skin CM hotspots better predict capture efficiency in sun-exposed normal skin than cSCC mutation hotspots, cSCC mutation hotspots can still design efficient sequencing targets in genomic areas where we do not have deep sequencing data on CM in normal skin. Our work provides a framework for helping design efficient, customized sequencing panels covering the genomic regions with the highest number of mutations. We expect the current algorithm to be a valuable tool for similar studies of other cancers. Citation Format: Sydney R. Grant, Megan E. Fitzgerald, Barbara A. Foster, Wendy J. Huss, Lei Wei, Gyorgy Paragh. Comparison of mutational burden hotspots in cutaneous squamous cell carcinoma and UV-exposed healthy skin for development of optimal targeted sequencing panels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1909.

Although recombinant human erythropoietin (rHuEpo) has revolutionized the treatment of anemia, recent clinical trials suggested that rHuEpo use may be associated with decreased survival in cancer patients. Although the expression of erythropoietin (Epo) receptor (EpoR) has been demonstrated in various human cancers, the effect of exogenous Epo on the growth and therapy resistance of EpoR-bearing tumor cells is unclear at present. In the current study, we examined the hypothesis that EpoR may contribute to tumor growth independent of Epo in A2780 human ovarian carcinoma cells. A2780 human ovarian carcinoma cells showed high levels of EpoR expression, but lacked expression of Epo mRNA and biologically active Epo protein under both normoxic and hypoxic conditions. Exogenous Epo did not stimulate EpoR-mediated signaling, proliferation, invasiveness, or resistance to cytotoxic drugs in A2780 cells. In contrast, specific inhibition of EpoR expression using a short hairpin RNA (shRNA) expression plasmid resulted in markedly reduced proliferation and invasiveness in vitro. In addition, inhibition of EpoR expression led to abrogated in vivo ovarian cancer cell growth in a tumor xenograft system and resulted in decreased EpoR signaling. Our findings suggest that EpoR may be constitutively active in some cancer cells in the absence of Epo and provide the first evidence for a potential role of an Epo-independent, EpoR-mediated pathway in the growth of some human cancers.

Although the earliest feature of disseminated disease in breast cancer is regional lymph node involvement, little is known about the mechanisms whereby cancer cells interact with lymphatic endothelial cells and enter the lymphatic system. We have previously reported that the extensive presence of retraction clefts in breast carcinomas highly significantly correlates with lymphatic tumor spread and predicts poor outcome, suggesting that retraction clefts are not just fixation artifacts, but real potential spaces that are exaggerated by tissue processing and may reflect an early stage of lymphatic invasion. In this study, we examined the correlation between the extent of retraction clefts and lymphangiogenesis, as assessed by lymphatic vessel density and vascular endothelial growth factor-C (VEGF-C) expression in a series of 256 early-stage breast carcinomas. The presence and extent of retraction clefts around tumor cell nests was determined by review of all hematoxylin- and eosin-stained tumor sections. Lymphatic vessels were detected by podoplanin immunohistochemistry and lymphatic vessel density was measured using the hot-spot method. The expression of VEGF-C in the tumor cells was determined by immunohistochemistry and analyzed semiquantitatively on a four-tiered scale. High levels of retraction clefts, peritumor lymphatic vessel density and VEGF-C expression at the invasive edge in breast carcinomas significantly correlated with tumor size, histological grade, lymphatic invasion and nodal metastasis. Breast carcinomas showing extensive retraction clefts (>20% of tumor volume) were found to have significantly higher lymphatic vessel density and VEGF-C expression levels compared to tumors without this feature. High retraction clefts, peritumor lymphatic vessel density and VEGF-C expression predicted poor outcome in breast carcinomas. Our results support the hypothesis that retraction clefts are real potential spaces that may represent ‘pre-lymphatic spaces’ facilitating initial lymphatic invasion and that growth factors secreted by the tumor cells may stimulate tumor-associated lymphangiogenesis by promoting the endothelialization of these ‘pre-lymphatic channels’.

Retraction artifact around tumor cell nests is a characteristic feature of invasive micropapillary carcinoma (IMPC), a special type of breast cancer commonly associated with nodal metastasis. We have recently reported that the extent of retraction artifact in usual invasive ductal carcinomas (IDC) is also a strong predictor of nodal metastasis. We examined whether the presence and extent of micropapillary features and retraction artifact in core needle biopsy of breast cancers can predict nodal metastasis in a prospective series of 47 IMPC and 424 IDC. Micropapillary features were present on core needle biopsy in 28 of 47 IMPC cases. Nodal metastases were found in 21 of 28 and 14 of 19 IMPC cases with and without micropapillary features present on core needle biopsy, respectively. Lymph node metastasis was significantly associated with the presence of micropapillary features, but not with its extent within these tumors. The presence of extensive retraction artifact in core needle biopsy samples of IDC also showed a significant association with nodal metastasis. Our results indicate that the presence of micropapillary features or extensive retraction artifact on core needle biopsy of breast carcinoma can predict nodal metastasis. Our results support the notion that the characteristic clear spaces separating the tumor cells from the stroma in IMPC and IDC of the breast are not a random artifactual phenomenon simply resulting from tissue fixation and processing, but rather they are likely related to altered tumor-stromal interactions, which might have an important role in lymphatic tumor spread.

Mixed connective tissue disease (MCTD) is a systemic inflammatory autoimmune disease. The connection between inflammatory measures and atherosclerosis in MCTD has not been described. Paraoxonase (PON) is known to have an antioxidant function. We evaluated lipid profiles and PON activity in patients with MCTD.Thirty-seven patients with MCTD were enrolled. Patients had taken no antihyperlipidemic drugs in the past 2 months. Thirty healthy individuals served as controls. The mean age of patients was 51.2 +/- 9.5 years; disease duration was 11.0 +/- 7.2 years. PON activity was determined with spectrophotometry, von Willebrand factor (vWF) antigen was investigated with the immunoturbidimetry method, and thrombomodulin and antiendothelial cell antibody (AECA) measurements were carried out by ELISA.PON activity in patients with MCTD was significantly lower than in the controls (patients 118.5 +/- 64.6 U/l, controls 188.0 +/- 77.6; p0.001). Arylesterase activity was significantly reduced in patients (p0.001). Reduction of PON activity showed a close correlation with the age of the patients, duration of the disease, and vascular events (eye, cardiac, cerebral). There was a close association between the low PON activity and endothelial cell activation markers (thrombomodulin, vWF, AECA).Our results indicate that in patients with MCTD there is an increased risk for atherosclerosis. In the development of atherosclerosis, besides the elevated levels of cholesterol and triglyceride, reduced PON concentrations and PON activity may play a crucial role.

Oral anticoagulants, in Hungary acenocoumarol being the one exclusively used, have a low therapeutic index and a high bleeding complication rate. The cytochrome P450 2C9 enzyme plays an important role in their metabolism.To investigate the influence of CYP2C9 polymorphism on the occurrence of bleeding complications related to acenocoumarol therapy.Genotyping of 421 patients (183 men and 238 women, mean age 66.2+/-11.8 years), who had been taking acenocoumarol for at least 6 months, was performed. Based on patient history and laboratory data, the correlations between genotype and acenocoumarol dose and bleeding complications were retrospectively analysed.In 145 patients bearing alleles with reduced activity (CYP2C9*2 and/or *3), the optimal dose of acenocoumarol was significantly (p0.001) lower than in patients with the wild type allele (2.12+/-0.96 mg/day and 2.90+/-1.46 mg/day, respectively). In comparison with wild type allele patients, the mean daily acenocoumarol dose was lower in the CYP2C9*2 group, and the lowest in *3 bearers. Although the occurrence of minor bleeding complications in patients with the variant allele was significantly (p0.005) higher (OR=1.99 [CI: 1.20-3.33]) than in other patients, there was no difference in major bleeding complications.Patients bearing CYP2C9 alleles with reduced enzymatic activity have a lower acenocoumarol requirement. In patients with CYP2C9*2 and *3 alleles the frequency of minor bleeding complications and the occurrence of high INR values were significantly higher, but there was no difference in the rate of major bleedings.

The aim of this study was to investigate the effect of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor fluvastatin (Flu) on angiotensin II (AII)–stimulated neutrophils of patients with hypercholesterolemia. Results suggest that a 6-week-long Flu administration completely counteracted the AII-induced increase in superoxide anion and leukotriene C4 production of the neutrophils of patients with hypercholesterolemia. However, the failure of signal processing through pertussis toxin–sensitive G protein, the increase in [Ca2+]i in membrane-bound protein kinase C activity, and the increase in neutrophil-bound cholesterol content were only partially restored by Flu. In addition, Flu had no effect on the increased membrane rigidity of the neutrophils of patients with hypercholesterolemia. To sum it up, Flu administration had a beneficial effect on AII-triggered reactive oxygen species generation; it resulted in partial restoration of signaling processes and of membrane composition, but membrane fluidity remained unchanged.

1. We recently described the sensory nitrergic nature of the hepatic insulin sensitizing substance (HISS) mechanism linked to postprandial activation of anterior hepatic plexus fibres in rabbits. This study is designed to assess the involvement of the sensory pathways in this mechanism. 2. Selective sensory denervation of the anterior hepatic plexus (AHP) was achieved by a 3-day perineurial treatment with 2% capsaicin solution in Wistar rats (230-250 g). After 1 week, hyperinsulinaemic (100 micro U kg(-1)) euglycaemic (5.5 mmol kg(-1)) glucose clamp studies were performed to estimate insulin sensitivity. 3. The rats with regional AHP sensory denervation exhibited a significantly decreased insulin sensitivity, that is, 9.1+/-1.0 mg kg(-1) min(-1) glucose reinstalled euglycaemia vs 13.3+/-1.9 mg kg(-1) min(-1) glucose (P0.01) in control rats. 4. Acute partial hepatic denervation by AHP cut was without effect on insulin sensitivity, whereas chronic hepatic denervation induced insulin resistance was similar to that achieved by regional AHP capsaicin treatment. 5. Intraportal administration of L-NAME (10 mg kg(-1)) decreased, whereas capsaicin (0.3 mg kg(-1) min(-1)) increased insulin sensitivity. Neither atropine (1 mg kg(-1)) nor acetylcholine (1-10 micro g mg min(-1)) produced any significant effect. In animals with preceding regional capsaicin desensitization, none of the pharmacological manoeuvres modified the resulting insulin-resistant state. 6. Cysteamine (200 mg kg(-1) s.c.) is known to cause functional somatostatin depletion-induced insulin resistance similar to that produced by either chronic partial hepatic denervation or perineurial AHP capsaicin desensitization. Intraportal capsaicin (0.3 mg kg(-1) min(-1)) was unable to modify insulin resistance achieved by cysteamine. 7. We conclude that capsaicin-sensitive sensory fibres play a crucial role in neurogenic insulin sensitization known as the HISS mechanism without involvement of anatomical reflex-mediated circuits. The results also suggest that HISS is identical to somatostatin of AHP sensory neural origin.