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Background Peritoneal metastases (PM) in colorectal cancer (CRC) are associated with therapy resistance and poor survival. Oxaliplatin monotherapy is widely applied in the intraperitoneal treatment of PM, but fails to yield clinical benefit. We aimed to identify the mechanism(s) underlying PM resistance to oxaliplatin and to develop strategies overcoming such resistance. Experimental design We generated a biobank consisting of 35 primary tumour regions and 59 paired PM from 12 patients. All samples were analysed by RNA sequencing. We also generated a series of PM-derived organoid (PMDO) cultures and used these to design and test strategies to overcome resistance to oxaliplatin. Results PM displayed various hallmarks of aggressive CRC biology. The vast majority of PM and paired primary tumours belonged to the Consensus Molecular Subtype 4 (CMS4). PMDO cultures were resistant to oxaliplatin and expressed high levels of glutamate-cysteine ligase (GCLC) causing detoxification of oxaliplatin through glutathione synthesis. Genetic or pharmacological targeting of GCLC sensitised PMDOs to a 1-h exposure to oxaliplatin, through increased platinum-DNA adduct formation. Conclusions These results link oxaliplatin resistance of colorectal PM to their CMS4 status and high reducing capacity. Inhibiting the reducing capacity of PM may be an effective strategy to overcome PM resistance to oxaliplatin.

Peritoneal metastases (PM) in colorectal cancer (CRC) are associated with therapy resistance and poor survival. Oxaliplatin monotherapy is widely applied in the intraperitoneal treatment of PM, but fails to yield clinical benefit. We aimed to identify the mechanism(s) underlying PM resistance to oxaliplatin and to develop strategies overcoming such resistance.We generated a biobank consisting of 35 primary tumour regions and 59 paired PM from 12 patients. All samples were analysed by RNA sequencing. We also generated a series of PM-derived organoid (PMDO) cultures and used these to design and test strategies to overcome resistance to oxaliplatin.PM displayed various hallmarks of aggressive CRC biology. The vast majority of PM and paired primary tumours belonged to the Consensus Molecular Subtype 4 (CMS4). PMDO cultures were resistant to oxaliplatin and expressed high levels of glutamate-cysteine ligase (GCLC) causing detoxification of oxaliplatin through glutathione synthesis. Genetic or pharmacological targeting of GCLC sensitised PMDOs to a 1-h exposure to oxaliplatin, through increased platinum-DNA adduct formation.These results link oxaliplatin resistance of colorectal PM to their CMS4 status and high reducing capacity. Inhibiting the reducing capacity of PM may be an effective strategy to overcome PM resistance to oxaliplatin.

IntroductionDespite its increasing use, first-line palliative systemic therapy alternated with electrostatic pressurised intraperitoneal aerosol chemotherapy with oxaliplatin (ePIPAC-OX), hereinafter referred to as first-line bidirectional therapy, has never been prospectively investigated in patients with colorectal peritoneal metastases (CPM). As a first step to address this evidence gap, the present study aims to assess the safety, feasibility, antitumour activity, patient-reported outcomes, costs and systemic pharmacokinetics of first-line bidirectional therapy in patients with isolated unresectable CPM.Methods and analysisIn this single-arm, phase II study in two Dutch tertiary referral centres, 20 patients are enrolled. Key eligibility criteria are a good performance status, pathologically proven isolated unresectable CPM, no previous palliative systemic therapy for colorectal cancer, no (neo)adjuvant systemic therapy ≤6 months prior to enrolment and no previous pressurised intraperitoneal aerosol chemotherapy (PIPAC). Patients receive three cycles of bidirectional therapy. Each cycle consists of 6 weeks first-line palliative systemic therapy at the medical oncologists’ decision (CAPOX-bevacizumab, FOLFOX-bevacizumab, FOLFIRI-bevacizumab or FOLFOXIRI-bevacizumab) followed by ePIPAC-OX (92 mg/m2) with an intraoperative bolus of intravenous leucovorin (20 mg/m2) and 5-fluorouracil (400 mg/m2). Study treatment ends after the third ePIPAC-OX. The primary outcome is the number of patients with—and procedures leading to—grade ≥3 adverse events (Common Terminology Criteria for Adverse Events V.5.0) up to 4 weeks after the last procedure. Key secondary outcomes include the number of bidirectional cycles in each patient, treatment-related characteristics, grade ≤2 adverse events, tumour response (histopathological, cytological, radiological, biochemical, macroscopic and ascites), patient-reported outcomes, systemic pharmacokinetics of oxaliplatin, costs, progression-free survival and overall survival.Ethics and disseminationThis study is approved by the Dutch competent authority, a medical ethics committee and the institutional review boards of both study centres. Results will be submitted for publication in peer-reviewed medical journals and presented to patients and healthcare professionals.Trial registration numberNL8303.

Introduction: Circulating tumor DNA (ctDNA) from plasma is a promising biomarker. Patients with metastatic colorectal cancer (CRC) often have high ctDNA levels compared to other cancer types. However, it is unclear whether this is also the case for patients with peritoneal metastases from CRC. Aim: To compare the propensity of ctDNA shedding by isolated liver metastases and isolated peritoneal metastases from CRC. Methods: Plasma was collected from 100 CRC patients (64% male, mean age of 60 [SD=10] years) with isolated unresectable liver metastasis (CRC-LM). Plasma and ascites were obtained from 20 CRC patients (60% male, mean age of 63 [SD=9.8] years) with isolated unresectable peritoneal metastases (CRC-PM). All liquid biopsies were obtained prior to treatment and cell-free DNA was isolated using the QIAsymphony (Qiagen, Germany) and assessed by droplet digital PCR (ddPCR; Bio-Rad, Hercules, USA). Patients with a KRAS or BRAF tumor tissue mutation were suited for plasma ctDNA ddPCR analysis, and were detected in 57 CRC-LM (57%) and 11 CRC-PM (55%) patients. The ability to shed ctDNA into the circulation was evaluated by comparing KRAS/BRAF mutation status in plasma and ascites with the mutation status based on tumor tissue. Regarding liquid biopsies, mutant allele fraction (MAF) and mutant copies per ml input (MTc/ml) were reported. Results: Tissue mutations could be confirmed in plasma in 93% of CRC-LM and only 20% of CRC-PM patients, whereas mutations were detected in ascites in 100% of CRC-PM patients. The MAF and MTc/ml were both significantly lower in CRC-PM plasma ctDNA (median MAF=0.28% and MTc/ml=21) compared to CRC-LM plasma ctDNA (median MAF=18.9% and MTc/ml= 1758; P Conclusion: To our knowledge, this is the first study showing a comprehensive comparison of tissue, blood and ascites derived genomic information in patients with CRC and extensive isolated peritoneal metastases. This study concludes that isolated peritoneal metastases from CRC is a malignancy with distinct clinical and biological characteristics, which should be taken into account when considering the clinical utility of ctDNA in different metastatic setting of CRC. To detect genomic alterations, the blood circulation is the preferred source of ctDNA in case of CRC liver metastases, whereas ascites offers an alternative to plasma in patients with peritoneal metastases, which might be suitable as diagnostic, prognostic, predictive or disease monitoring biomarker. Citation Format: Iris van 't Erve, Koen P. Rovers, Alexander Constantinides, Karen Bolhuis, Emma C. Wassenaar, Robin J. Lurvink, Clément J. Huysentruyt, Petur Snaebjornsson, Daan van den Broek, Tineke E. Buffart, Niels F. Kok, Gerrit A. Meijer, Cornelis J. Punt, Onno Kranenburg, Ignace H. de Hingh, Remond J. Fijneman. Limited release of circulating tumor DNA into the systemic circulation by peritoneal metastases from colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 708.

IntroductionCytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) has become standard of care for patients with peritoneal metastases of colorectal origin with a low/moderate abdominal disease load. In case of a peritoneal cancer index (PCI) score >20, CRS-HIPEC is not considered to be beneficial. Patients with a PCI >20 are currently offered palliative systemic chemotherapy. Previous studies have shown that systemic chemotherapy is less effective against peritoneal metastases than it is against haematogenous spread of colorectal cancer. It is suggested that patients with peritoneal metastases may benefit from the addition of intraperitoneal chemotherapy to systemic chemotherapy. Aim of this study is to establish the maximum tolerated dose of intraperitoneal irinotecan, added to standard of care systemic therapy for colorectal cancer. Secondary endpoints are to determine the safety and feasibility of this treatment and to establish the pharmacokinetic profile of intraperitoneally administered irinotecan.Methods and analysisThis phase I, ‘3+3’ dose-escalation, study is performed in two Dutch tertiary referral centres. The study population consists of adult patients with extensive peritoneal metastases of colorectal origin who have a good performance status and no extra-abdominal metastases. According to standard work-up for CRS-HIPEC, patients will undergo a diagnostic laparoscopy to score the PCI. In case of a PCI >20, a peritoneal access port will be placed in the abdomen of the patient. Through this port we will administer intraperitoneal irinotecan, in combination with standard systemic treatment consisting of 5-fluorouracil/leucovorin with oxaliplatin and the targeted agent bevacizumab. Therapy consists of a maximum of 12 cycles 2-weekly.Ethics and disseminationThis study protocol is approved by a research medical ethics committee (Rotterdam, Netherlands) and the Dutch Competent Authority (CCMO, The Hague, Netherlands). The results of this trial will be submitted for publication in a peer-reviewed scientific journal.Trail registration numberNL6988 and NL2018-000479-33; Pre-results.

IntroductionRepetitive electrostatic pressurised intraperitoneal aerosol chemotherapy with oxaliplatin (ePIPAC-OX) is offered as a palliative treatment option for patients with isolated unresectable colorectal peritoneal metastases (PM) in several centres worldwide. However, little is known about its feasibility, safety, tolerability, efficacy, costs and pharmacokinetics in this setting. This study aims to explore these parameters in patients with isolated unresectable colorectal PM who receive repetitive ePIPAC-OX as a palliative monotherapy.Methods and analysisThis multicentre, open-label, single-arm, phase II study is performed in two Dutch tertiary referral hospitals for the surgical treatment of colorectal PM. Eligible patients are adults who have histologically or cytologically proven isolated unresectable PM of a colorectal or appendiceal carcinoma, a good performance status, adequate organ functions and no symptoms of gastrointestinal obstruction. Instead of standard palliative treatment, enrolled patients receive laparoscopy-controlled ePIPAC-OX (92 mg/m2body surface area (BSA)) with intravenous leucovorin (20 mg/m2BSA) and bolus 5-fluorouracil (400 mg/m2BSA) every 6 weeks. Four weeks after each procedure, patients undergo clinical, radiological and biochemical evaluation. ePIPAC-OX is repeated until disease progression, after which standard palliative treatment is (re)considered. The primary outcome is the number of patients with major toxicity (grade ≥3 according to the Common Terminology Criteria for Adverse Events v4.0) up to 4 weeks after the last ePIPAC-OX. Secondary outcomes are the environmental safety of ePIPAC-OX, procedure-related characteristics, minor toxicity, postoperative complications, hospital stay, readmissions, quality of life, costs, pharmacokinetics of oxaliplatin, progression-free survival, overall survival, and the radiological, histopathological, cytological, biochemical and macroscopic tumour response.Ethics and disseminationThis study is approved by an ethics committee, the Dutch competent authority and the institutional review boards of both study centres. Results are intended for publication in peer-reviewed medical journals and for presentation to patients, healthcare professionals and other stakeholders.Trial registration numberNCT03246321, Pre-results;ISRCTN89947480, Pre-results; NTR6603, Pre-results; EudraCT: 2017-000927-29, Pre-results.

Eur J Clin Invest 2012; 42 (11): 12351243 Abstract Background Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a novel cardiovascular risk marker, which is predominantly complexed to apolipoprotein (apo) B-containing lipoproteins in human plasma. As increasing dietary sodium intake may decrease plasma apoB-containing lipoproteins, we tested whether a sodium challenge lowers plasma Lp-PLA2 mass, as well as the levels of apoB-containing lipoprotein particles carrying Lp-PLA2 (apoB-Lp-PLA2), employing a newly developed enzyme-linked immunosorbent assay. Materials and methods In 45 women and 31 men (mean age 44 +/- 14 years), plasma Lp-PLA2 mass (turbidimetric immunoassay), the level of apoB-Lp-PLA2, expressed in apoB concentration and lipoproteins were measured in response to a 3-day challenge with 9 g sodium chloride tablets daily. Results Urinary sodium excretion increased from 165 +/- 60 to 321 +/- 70 mmol/24 h (P

Context:Plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) predicts incident cardiovascular disease and is associated preferentially with negatively charged apolipoprotein B-containing lipoproteins. The plasma cholesteryl ester transfer (CET) process, which contributes to low high-density lipoprotein cholesterol and small, dense low-density lipoproteins, is affected by the composition and concentration of apolipoprotein B-containing cholesteryl ester acceptor lipoproteins.Objective:We tested relationships of CET with Lp-PLA2 in subjects with and without metabolic syndrome (MetS).Design and Setting:In 68 subjects with MetS and 74 subjects without MetS, plasma Lp-PLA2 mass, cholesterol esterification (EST), lecithin:cholesterol acyltransferase (LCAT) activity level, CET, CET protein (CETP) mass, and lipoproteins were measured.Results:EST, LCAT activity, CET (P < 0.001 for all), and CETP (P = 0.030) were increased, and Lp-PLA2 was decreased (P = 0.043) in MetS. CET was correlated positively with Lp-PLA2 in subjects with and without MetS (P < 0.05 for both). EST and LCAT activity were unrelated to Lp-PLA2, despite a positive correlation between EST and CET (P < 0.001). After controlling for age, sex, and diabetes status, CET was determined by Lp-PLA2 in the whole group (β = 0.245; P < 0.001), and in subjects with (β = 0.304; P = 0.001) and without MetS (β = 0.244; P = 0.006) separately, independently of triglycerides and CETP.Conclusions:Plasma CET is related to Lp-PLA2 in subjects with and without MetS. The process of CET, but not EST, may be influenced by Lp-PLA2. These findings provide a rationale to evaluate whether maneuvers that inhibit Lp-PLA2 will reduce CET, and vice versa to document effects of CETP inhibition on Lp-PLA2.

Background A recent meta-analysis showed that both plasma lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) mass and activity independently predict cardiovascular events. Notably, Lp-PLA(2) activity but not mass was found to be a determinant of cardiovascular outcome in type 2 diabetes mellitus. We questioned whether relationships of carotid intima media thickness (IMT), a measure of subclinical atherosclerosis, with Lp-PLA(2) mass differ between diabetic and nondiabetic subjects. Materials and methods Relationships of IMT with plasma Lp-PLA(2) mass (turbidimetric immunoassay) were compared in 74 patients with type 2 diabetes and in 64 nondiabetic subjects. Results IMT was increased (P=0.016), but plasma Lp-PLA(2) mass was decreased in patients with diabetes compared to nondiabetic subjects (277 +/- 66 vs. 327 +/- 62 mu g L-1, P Conclusions Plasma Lp-PLA(2) may relate to early stages of atherosclerosis development. In diabetes mellitus, in contrast, the association of IMT with plasma Lp-PLA(2) mass is abolished, which could be partly ascribed to redistribution of Lp-PLA(2) mass from apolipoprotein B-containing lipoproteins towards HDL. These findings raise questions about the usefulness of plasma Lp-PLA(2) mass measurement as a marker of subclinical atherosclerosis in type 2 diabetes mellitus.

Objective Plasma lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) levels predict incident cardiovascular disease, impacting Lp-PLA 2 as an emerging therapeutic target. We determined Lp-PLA 2 responses to statin and fibrate administration in type 2 diabetes mellitus, and assessed relationships of changes in Lp-PLA 2 with subclinical inflammation and lipoprotein characteristics. Methods A placebo-controlled cross-over study (three 8-week treatment periods with simvastatin (40 mg daily), bezafibrate (400 mg daily) and their combination) was carried out in 14 male type 2 diabetic patients. Plasma Lp-PLA 2 mass was measured by turbidimetric immunoassay. Results Plasma Lp-PLA 2 decreased (− 21 ± 4%) in response to simvastatin ( p 2 during combined treatment (− 17 ± 3%, p 2 changes during the 3 active lipid lowering treatment periods were related positively to baseline levels of high sensitive C-reactive protein, non-HDL cholesterol, triglycerides, the total cholesterol/HDL cholesterol ratio and less LDL electronegativity ( p p 2 ( p 2 responses correlated inversely with changes in non-HDL cholesterol, triglycerides and the total cholesterol/HDL cholesterol ratio during treatment ( p p Conclusions In type 2 diabetes mellitus, plasma Lp-PLA 2 is likely to be lowered by statin treatment only. Enhanced subclinical inflammation and more severe dyslipidemia may predict diminished LpPLA 2 responses during lipid lowering treatment, which in turn appear to be quantitatively dissociated from decreases in apolipoprotein B lipoproteins. Conventional lipid lowering treatment may be insufficient for optimal LpPLA 2 lowering in diabetes mellitus.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) ) is a novel cardiovascular risk marker, which is predominantly complexed to apolipoprotein (apo) B-containing lipoproteins in human plasma. As increasing dietary sodium intake may decrease plasma apoB-containing lipoproteins, we tested whether a sodium challenge lowers plasma Lp-PLA(2) mass, as well as the levels of apoB-containing lipoprotein particles carrying Lp-PLA(2) (apoB-Lp-PLA(2) ), employing a newly developed enzyme-linked immunosorbent assay.In 45 women and 31 men (mean age 44 ± 14 years), plasma Lp-PLA(2) mass (turbidimetric immunoassay), the level of apoB-Lp-PLA(2) , expressed in apoB concentration and lipoproteins were measured in response to a 3-day challenge with 9 g sodium chloride tablets daily.Urinary sodium excretion increased from 165 ± 60 to 321 ± 70 mmol/24 h (P0.001) after salt loading. Plasma Lp-PLA(2) mass decreased from 618 (493-719) to 588 (465-698) μg/L (P0.001), and apoB-Lp-PLA(2) decreased from 0.276 (0.200-0.351) to 0.256 (0.189-0.328) g LDL protein/L (P=0.004) in response to the sodium challenge together with decreases in plasma total cholesterol, nonhigh-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, apolipoprotein B and the total cholesterol/HDL cholesterol ratio (P0.01 for all). Changes in plasma Lp-PLA(2) mass were correlated positively with changes in total cholesterol, LDL cholesterol and non-HDL cholesterol (r=0.260-0.276, P0.05 to P0.02), whereas changes in apoB-Lp-PLA(2) were correlated positively with changes in non-HDL cholesterol and in the total cholesterol/HDL cholesterol ratio (r=0.232-0.385, P0.05-0.01).Both plasma Lp-PLA(2) mass levels and apoB-Lp-PLA(2) decrease in response to a short-term oral sodium challenge.

Plasma lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) levels predict incident cardiovascular disease, impacting Lp-PLA(2) as an emerging therapeutic target. We determined Lp-PLA(2) responses to statin and fibrate administration in type 2 diabetes mellitus, and assessed relationships of changes in Lp-PLA(2) with subclinical inflammation and lipoprotein characteristics.A placebo-controlled cross-over study (three 8-week treatment periods with simvastatin (40 mg daily), bezafibrate (400mg daily) and their combination) was carried out in 14 male type 2 diabetic patients. Plasma Lp-PLA(2) mass was measured by turbidimetric immunoassay.Plasma Lp-PLA(2) decreased (-21 ± 4%) in response to simvastatin (p0.05 from baseline and placebo), but was unaffected by bezafibrate (1 ± 5%). The drop in Lp-PLA(2) during combined treatment (-17 ± 3%, p0.05) was similar compared to that during simvastatin alone. The Lp-PLA(2) changes during the 3 active lipid lowering treatment periods were related positively to baseline levels of high sensitive C-reactive protein, non-HDL cholesterol, triglycerides, the total cholesterol/HDL cholesterol ratio and less LDL electronegativity (p0.02 to p0.01), and inversely to baseline Lp-PLA(2) (p0.01). LpPLA(2) responses correlated inversely with changes in non-HDL cholesterol, triglycerides and the total cholesterol/HDL cholesterol ratio during treatment (p0.05 to p0.02).In type 2 diabetes mellitus, plasma Lp-PLA(2) is likely to be lowered by statin treatment only. Enhanced subclinical inflammation and more severe dyslipidemia may predict diminished LpPLA(2) responses during lipid lowering treatment, which in turn appear to be quantitatively dissociated from decreases in apolipoprotein B lipoproteins. Conventional lipid lowering treatment may be insufficient for optimal LpPLA(2) lowering in diabetes mellitus.

Background and Aims Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) is a pro-atherogenic phospholipase A 2 , which is predominantly complexed to low-density lipoprotein (LDL) particles. Proprotein convertase subtilisin-kexin type 9 (PCSK9) provides a key step in LDL metabolism by stimulating LDL receptor degradation. We determined relationships between plasma PCSK9 and Lp-PLA 2 mass. Methods Lp-PLA 2 mass (turbidimetric immunoassay), PCSK9 (enzyme-linked immunosorbent assay) and (apo) lipoproteins were measured in 53 nondiabetic subjects (27 women) with body mass index 2 . Results Lp-PLA 2 and PCSK9 levels were both correlated positively with LDL cholesterol and non-high-density lipoprotein (HDL) cholesterol (r = 0.330 to r = 0.382, p ≤0.02). Remarkably, Lp-PLA 2 was inversely related to PCSK9 (r = −0.388, p = 0.004). The Lp-PLA 2 /apolipoprotein B ratio, as a measure of the Lp-PLA 2 content in apolipoprotein B-containing lipoproteins, was also inversely correlated with PCSK9 (r = −0.575, p 2 ( p = 0.023) and the Lp-PLA 2 /apolipoprotein B ratio ( p = 0.001) with PCSK9 levels remained significant after controlling for age, gender, triglycerides and HDL cholesterol. Conclusions Despite increasing effects on LDL cholesterol, higher PCSK9 levels are unlikely to confer impaired Lp-PLA 2 metabolism. We propose to evaluate the possible influence of PCSK9 inhibiting strategies on Lp-PLA 2 regulation and vice versa to determine effects of Lp-PLA 2 inhibitors on the PCSK9 pathway.

A recent meta-analysis showed that both plasma lipoprotein-associated phospholipase A(2) (Lp-PLA(2) ) mass and activity independently predict cardiovascular events. Notably, Lp-PLA(2) activity but not mass was found to be a determinant of cardiovascular outcome in type 2 diabetes mellitus. We questioned whether relationships of carotid intima media thickness (IMT), a measure of subclinical atherosclerosis, with Lp-PLA(2) mass differ between diabetic and nondiabetic subjects.Relationships of IMT with plasma Lp-PLA(2) mass (turbidimetric immunoassay) were compared in 74 patients with type 2 diabetes and in 64 nondiabetic subjects.IMT was increased (P=0·016), but plasma Lp-PLA(2) mass was decreased in patients with diabetes compared to nondiabetic subjects (277±66 vs. 327±62μgL(-1) , P0·001). In nondiabetic subjects, IMT was correlated positively with Lp-PLA(2) (r=0·325, P0·009); multiple linear regression analysis confirmed an independent association of IMT with Lp-PLA(2) (ß=0·192, P=0·048). In contrast, IMT was unrelated to Lp-PLA(2) in patients with diabetes (r=0·021, P=0·86), and the relationship of IMT with Lp-PLA(2) was different in diabetic and control subjects (P0·001). The relationship of Lp-PLA(2) with the total cholesterol/high-density lipoprotein (HDL) cholesterol ratio also differed between diabetic and nondiabetic subjects (P0·001).Plasma Lp-PLA(2) may relate to early stages of atherosclerosis development. In diabetes mellitus, in contrast, the association of IMT with plasma Lp-PLA(2) mass is abolished, which could be partly ascribed to redistribution of Lp-PLA(2) mass from apolipoprotein B-containing lipoproteins towards HDL. These findings raise questions about the usefulness of plasma Lp-PLA(2) mass measurement as a marker of subclinical atherosclerosis in type 2 diabetes mellitus.