Collagen biology and non-invasive biomarkers of liver fibrosis.

There is an unmet need for high-quality liquid biomarkers that can safely and reproducibly predict the stage of fibrosis and the outcomes of chronic liver disease (CLD). The requirement for such markers has intensified because of the high global prevalence of diseases such as non-alcoholic fatty liver disease (NAFLD). In particular, there is a need for diagnostic and prognostic tools, as well as predictive biomarkers that reflect the efficacy of interventions, as described by the BEST criteria (Biomarkers, EndpointS, and other Tools Resource). This review covers the various liver collagens, their functional role in tissue homeostasis and delineates the common nomenclature for biomarkers based on BEST criteria. It addresses the common confounders affecting serological biomarkers, and describes defined collagen epitope biomarkers that originate from the dynamic processes of extracellular matrix (ECM) remodelling during liver injury.

Pegbelfermin (BMS-986036), a PEGylated fibroblast growth factor 21 analogue, in patients with non-alcoholic steatohepatitis: a randomised, double-blind, placebo-controlled, phase 2a trial.

BACKGROUND: Pegbelfermin (BMS-986036), a PEGylated human fibroblast growth factor 21 (FGF21) analogue, has previously been shown to improve markers of metabolism and liver fibrosis in obese patients with type 2 diabetes. In this phase 2a study, we aimed to evaluate the safety and efficacy of pegbelfermin in patients with non-alcoholic steatohepatitis. METHODS: In this multicentre, randomised, double-blind, placebo-controlled, parallel-group, phase 2a study, we recruited adults (aged 21-75 years) with a body-mass index of at least 25 kg/m2, biopsy-confirmed non-alcoholic steatohepatitis (fibrosis stage 1-3), and a hepatic fat fraction of at least 10% when assessed by magnetic resonance imaging-proton density fat fraction. These patients were enrolled at 17 medical centres in the USA. Eligible patients were stratified by type 2 diabetes status and they were randomly assigned (1:1:1) by a computer-based system to receive subcutaneous injections of placebo once a day, 10 mg pegbelfermin once a day, or 20 mg pegbelfermin once a week, all for 16 weeks. Participants, the study team administering treatment, and investigators analysing outcomes (who were independent of the study team and had no further involvement) were masked to treatment groups. The primary outcomes were safety and the absolute change in hepatic fat fraction after 16 weeks of treatment. All patients who were randomly assigned to groups and received the study drug or placebo were included in the primary analyses. This trial was registered with ClinicalTrials.gov, number NCT02413372. FINDINGS: Between May 12, 2015, and Aug 4, 2016, 184 overweight or obese patients with non-alcoholic steatohepatitis were screened for study inclusion. Of these, 95 (52%) patients were excluded because they no longer met study criteria and 80 (43%) patients entered the placebo lead-in phase. After further exclusions, 75 (94%) patients were randomly assigned to groups, received at least one dose of treatment (25 patients to receive 10 mg pegbelfermin once a day; 24 patients to receive 20 mg pegbelfermin once a week, and 26 patients to receive placebo), and were included in the primary analysis. A prespecified interim analysis at week 8 showed a greater than expected change in the primary outcome and supported early closing of patient enrolment, since this analysis indicated that the full planned sample size was not needed. We observed a significant decrease in absolute hepatic fat fraction in the group receiving 10 mg pegbelfermin daily (-6·8% vs -1·3%; p=0·0004) and in the group receiving 20 mg pegbelfermin weekly (-5·2% vs -1·3%; p=0·008) compared with the placebo group. Most adverse events were mild; the most common events were diarrhoea in eight (16%) of 49 patients treated with pegbelfermin and two (8%) of 26 patients treated with placebo and nausea in seven (14%) patients treated with pegbelfermin and two (8%) patients treated with placebo. There were no deaths, discontinuations due to adverse events, or treatment-related serious adverse events. INTERPRETATION: Treatment with subcutaneously administered pegbelfermin for 16 weeks was generally well tolerated and significantly reduced hepatic fat fraction in patients with non-alcoholic steatohepatitis. Further study of pegbelfermin is warranted in patients with non-alcoholic steatohepatitis. Additional studies that use liver biopsies would allow for the assessment of pegbelfermin's effects on liver histology. Moreover, further studies should allow assessments of the safety and effectiveness of pegbelfermin in a larger number of patients. FUNDING: Bristol-Myers Squibb.

Pegbelfermin (BMS-986036), PEGylated FGF21, in Patients with Obesity and Type 2 Diabetes: Results from a Randomized Phase 2 Study.

OBJECTIVE: Obesity and type 2 diabetes mellitus (T2DM) are risk factors for nonalcoholic fatty liver disease, including nonalcoholic steatohepatitis. This study assessed pegbelfermin (BMS-986036), recombinant PEGylated human fibroblast growth factor 21 (FGF21), in patients with obesity and T2DM predisposed to fatty liver. METHODS: In this randomized, double-blind, placebo-controlled study, patients with T2DM and BMI of 30 to 50 kg/m2 received subcutaneous pegbelfermin (1, 5, or 20 mg daily or 20 mg weekly; n = 96) or placebo (n = 24) for 12 weeks. Primary end points were safety, tolerability, and change in HbA1c. Additional end points included insulin sensitivity, lipids, adiponectin, and disease progression biomarkers. RESULTS: There were no significant effects of pegbelfermin versus placebo on HbA1c. Pegbelfermin 20 mg/d significantly improved high-density lipoprotein cholesterol (P = 0.015) and triglycerides (P = 0.037). All pegbelfermin regimens significantly increased adiponectin levels; 20-mg daily and weekly regimens decreased serum PRO-C3. Most adverse events were mild; the most frequent adverse events were injection-site bruising and diarrhea. CONCLUSIONS: Twelve-week pegbelfermin treatment did not impact HbA1c concentrations, but QW and higher daily doses were associated with improved metabolic parameters and fibrosis biomarkers in patients with obesity and T2DM predisposed to fatty liver. These results support evaluation of pegbelfermin in patients with obesity-related metabolic diseases (e.g., nonalcoholic steatohepatitis).

Randomized, Double-Blind, Placebo-Controlled, Phase 2 Trial of BMS-986020, a Lysophosphatidic Acid Receptor Antagonist for the Treatment of Idiopathic Pulmonary Fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) causes irreversible loss of lung function. The lysophosphatidic acid receptor 1 (LPA1) pathway is implicated in IPF etiology. Safety and efficacy of BMS-986020, a high-affinity LPA1 antagonist, was assessed vs placebo in a phase 2 study in patients with IPF. METHODS: IM136003 was a phase 2, parallel-arm, multicenter, randomized, double-blind, placebo-controlled trial. Adults with IPF (FVC, 45%-90%; diffusing capacity for carbon monoxide, 30%-80%) were randomized to receive placebo or 600 mg BMS-986020 (once daily [qd] or bid) for 26 weeks. The primary end point was rate of change in FVC from baseline to week 26. RESULTS: Of 143 randomized patients, 108 completed the 26-week dosing phase. Thirty-five patients discontinued prematurely. Patient baseline characteristics were similar between treatment groups (placebo: n = 47; 600 mg qd: n = 48; 600 mg bid: n = 48). Patients treated with BMS-986020 bid experienced a significantly slower rate of decline in FVC vs placebo (-0.042 L; 95% CI, -0.106 to -0.022 vs -0.134 L; 95% CI, -0.201 to -0.068, respectively; P = .049). Dose-related elevations in hepatic enzymes were observed in both BMS-986020 treatment groups. The study was terminated early because of three cases of cholecystitis that were determined to be related to BMS-986020 after unblinding. CONCLUSIONS: BMS-986020 600 mg bid treatment for 26 weeks vs placebo significantly slowed the rate of FVC decline. Both regimens of BMS-986020 were associated with elevations in hepatic enzymes. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01766817; URL: www.clinicaltrials.gov.

An Evaluation of the Collagen Fragments Related to Fibrogenesis and Fibrolysis in Nonalcoholic Steatohepatitis.

Fibrosis, resulted from the imbalance of fibrogenesis and fibrolysis, is a key readout of disease progression in nonalcoholic steatohepatitis (NASH) and reflects mortality risk. Non-invasive biomarkers capable of diagnosing fibrosis stages and monitoring fibrosis changes in NASH patients are urgently needed. This study is to evaluate collagen formation and degradation biomarkers, reflective of fibrogenesis or fibrolysis, in patients with biopsy proven NASH. Collagen formation biomarker PRO-C3 and PRO-C6 levels were significantly higher in patients with advanced fibrosis stage 3-4 than those with fibrosis stage 0-2. Elevated PRO-C3 levels were also associated with severe lobular inflammation and ballooning, but not with steatosis. Multivariate logistic regression analysis identified PRO-C3 and PRO-C6 to be independently related to fibrosis stage. PRO-C3 showed similar performance to identify patients with advanced fibrosis in discovery and validation cohorts. Furthermore, in a longitudinal study cohort with paired biopsies, mean PRO-C3 increased with worsening of fibrosis and decreased with fibrosis improvement. The results suggest that PRO-C3 may be a potentially useful biomarker in identifying patients with advanced fibrosis and active fibrogenesis, as well as in assessing changes in fibrosis over time. It is worthy of further evaluation to confirm its diagnostic value and clinical utility.

Phentolamine continuous infusion in a patient with pheochromocytoma.

PURPOSE: Use of continuous phentolamine infusion therapy for management of serious cardiovascular complications during adrenalectomy for pheochromocytoma is reported. SUMMARY: In preparation for surgical resection of a pheochromocytoma, a 38-year-old woman received outpatient oral therapy with the α-adrenergic-receptor blocker phenoxybenzamine for 25 days with the goal of reducing cardiovascular risks associated with catecholamine surge during surgery. Due to inappropriate dosage adjustment, however, outpatient phenoxybenzamine therapy did not achieve adequate α-adrenergic-receptor blockade; during the laparoscopic resection procedure, the woman developed severe hypertension, leading to cardiac arrest and discontinuation of the operation. After resuscitative measures, the patient was admitted to the surgical intensive care unit for mechanical ventilation, medical management (including intermittent bolus injections of phentolamine and a continuous i.v. infusion of esmolol for control of blood pressure and heart rate), and hemodynamic monitoring; despite those measures, cardiovascular instability persisted during the immediate postoperative period. The day after the abortive surgery attempt, a continuous infusion of phentolamine mesylate (1 mg/hr, adjusted hourly to achieve the blood pressure target) was initiated. Four days after initiation of continuous phentolamine infusion, the patient was deemed to be hemodynamically stable, and the surgery was successfully performed. CONCLUSION: A continuous infusion of phentolamine was used in a patient with pheochromocytoma to control perioperative hypertensive episodes during surgical adrenalectomy.

Thyrotoxicosis due to ectopic lateral thyroid tissue presenting 5 years after total thyroidectomy.

OBJECTIVE: To describe a patient who, 5 years after total thyroidectomy to treat Graves disease, presented with thyrotoxicosis due to nonmalignant lateral ectopic thyroid tissue. METHOD: We describe the laboratory, imaging, and physical findings of the study patient and review the relevant literature. RESULTS: A 32-year-old white woman with a history of Graves disease presented with recurrent hyperthyroidism 5 years after total thyroidectomy. A radioactive iodine scan was performed, which revealed elevated uptake (40%) and positive imaging in the left mid-neck. Ultrasonography examination of the neck confirmed the absence of any thyroid tissue within the thyroid bed, but documented 2 nodular, hypoechoic left upper-neck masses with punctuate hyperlucency. Contrast-enhanced computed tomography was performed to precisely localize the nodules, which were excised surgically via selective neck dissection. Histopathologic examination revealed chronic lymphocytic inflammatory infiltrate with focal thyroid hyperplasia and papillary infoldings and no evidence of malignancy. CONCLUSIONS: To our knowledge, this represents the first report of ectopic benign thyroid tissue as the sole cause of hyperthyroid symptoms, and this entity should be considered in patients who have undergone thyroidectomy and have persistent hyperthyroidism.

An atlas improves interobserver agreement regarding application of the ISCD vertebral body exclusion criteria.

Coexisting conditions such as osteoarthritis and compression fracture may spuriously elevate the dual-energy X-ray absorptiometry (DXA)-measured lumbar spine bone mass. To improve the diagnostic utility of lumbar spine DXA to diagnose osteoporosis, the International Society for Clinical Densitometry (ISCD) suggests excluding vertebrae affected by focal structural anomalies or unusual T-score discrepancies. However, we previously demonstrated only moderate agreement between physicians regarding vertebral body exclusion. We hypothesized that an atlas containing examples of vertebrae to exclude would improve interobserver agreement. Subsequently, we developed an interactive web-based atlas of lumbar spine DXA images with options to exclude vertebrae and compare one's answers to those derived by group consensus. Before and after review of the atlas, 5 ISCD-certified physicians applied the exclusion criteria to 90 DXA scans, recording the indications for vertebral exclusion on a standardized worksheet. After development and review of the atlas, interobserver agreement regarding vertebral body exclusion improved significantly (p<0.0001). We plotted the deviation of each physician's reported T-score vs the mean T-score for each of 90 scans, and demonstrated that the scatter from the mean is decreased after atlas review. Furthermore, correlations in T-score improved in 7 of 10 physician pairs after atlas review. We conclude that an interactive atlas promotes uniform lumbar spine DXA interpretation.

Radioguided parathyroidectomy for recurrent parathyroid cancer.

We report a case of radioguided parathyroidectomy in a patient with parathyroid carcinoma. A 61-year-old woman presented to our center with persistent hypercalcemia (17.2 mg/dL) and hyperparathyroidism (PTH=324 pg/mL) following her second neck resection for recurrent parathyroid carcinoma at an outside facility. Her elevated serum calcium had not responded to treatment with intravenous bisphosphonates, furosemide, or calcitonin. Calcimemetic therapy (Cinacalcet) was effective but had to be discontinued due to GI intolerance. She requested a second opinion at our center after being referred for palliative radiation therapy for presumed inoperable disease. On presentation, she remained symptomatic with bone and joint pain, diffuse abdominal pain and fatigue. Repeat technetium-99m sestamibi (Tc-99m sestamibi) scintigraphy showed a faint area of uptake near the right clavicular head, adjacent to the site of her previous resections. With the intraoperative guidance of a hand-held gamma probe, a 2 cm recurrent parathyroid carcinoma was located and successfully excised. Intraoperative PTH levels confirmed surgical cure of this previously undetected foci of disease. The use of radioguidance and intraoperative PTH monitoring were the keys to a successful resection, and our patient remains disease free with 17 months of follow-up.

Traumatic fracture in a healthy man: benign or pathologic?

OBJECTIVE: To describe the challenge of determining the correct diagnosis in a healthy adult male patient with a recent femoral fracture and a history of multiple bone fractures. METHODS: We present clinical, radiologic, laboratory, and histopathologic details in a patient with a history of recurrent fractures associated with minimal trauma. Moreover, the various types of osteopetrosis are reviewed. RESULTS: A 34-year-old African American man was in his usual state of good health when he fell hard on concrete. Immediately after the fall, he was able to bear weight, although pain prompted him to seek medical care. Besides a personal history of multiple fractures, he had no other medical problems. He had never smoked, denied illicit drug use, and had no family history of bone disorders or recurrent fractures. Findings on physical examination were unremarkable. Radiography disclosed an incomplete femoral fracture and osteosclerosis. Bone survey revealed diffuse, symmetric osteosclerosis of both the axial and the appendicular skeleton. The long bones showed areas of almost complete obliteration of the medullary canal, along with prominent hyperostosis. Additionally, a "bone-within-bone" appearance to the thickened endosteum was noted. A bone scan demonstrated numerous areas of symmetric radiotracer uptake. Laboratory analyses were unremarkable, including a complete blood cell count, electrolytes, serum protein electrophoresis, thyrotropin, and parathyroid hormone. Total alkaline phosphatase was mildly elevated at 162 U/L (normal range, 35 to 130). Seven needles were broken during attempts to perform a bone biopsy. Histologic examination showed normal bone marrow with "woven" bone and areas of primary spongiosa within mature osteoid. Autosomal dominant osteopetrosis type 2 was diagnosed on the basis of his clinical presentation and the radiologic and pathologic findings. CONCLUSION: The preliminary diagnosis for this patient's condition was Paget's disease, and determining the correct diagnosis of osteopetosis prevented the administration of inappropriate therapy. In addition, this case report reminds the clinician that genetic disease may manifest in adulthood.

Intimal estrogen receptor (ER)beta, but not ERalpha expression, is correlated with coronary calcification and atherosclerosis in pre- and postmenopausal women.

BACKGROUND: Controversy exists over the association of estrogen and cardiovascular disease. Estrogen receptors (ERs) alpha and beta are expressed in the endothelial cells and vascular smooth muscle cells (VSMCs) of many arteries, but the relative importance of ERalpha or ERbeta in mediating the vascular response to estrogens is not well defined, particularly in humans. We have shown previously that postmenopausal women receiving hormone therapy (HT) had lower mean coronary artery calcium, plaque area, and calcium-to-plaque ratio compared with untreated women. In this study, we examined coronary artery ERalpha and ERbeta expression in pre- and postmenopausal women as a function of plaque area, calcium area, calcium-to-plaque ratio, and estrogen status. METHODS: Coronary arteries were obtained at autopsy from a total of 55 women: nine premenopausal women, 13 postmenopausal women on HT and 33 untreated postmenopausal women (non-HT). Coronary calcification was quantified by contact microradiography, and atherosclerotic plaque area was measured histologically. Coronary artery cross-sections were immunostained for ERalpha and ERbeta, and the amount of receptors was estimated semiquantitatively in each arterial wall layer (intima, adventitia, and media). Double immunofluorescence was used to colocalize ERalpha and ERbeta with smooth muscle actin, a marker of VSMCs. RESULTS: ERbeta and ERalpha were expressed in all artery wall layers, but most avidly in the media (P = 0.001), and colocalized with VSMCs. ERbeta expression exceeded ERalpha expression in all wall layers (P < 0.001) and was adjacent to areas of calcium deposition. ERbeta expression in the intimal layer correlated with calcium content, plaque area, and calcium-to-plaque ratio (all P < 0.01) and tended to be greater in non-HT than in HT women (P = 0.06). ERalpha expression did not vary significantly among groups, nor did it correlate with calcium content, plaque area or calcium-to-plaque ratio. Expression of ERalpha but not ERbeta declined with age (P < 0.01) in HT women only. Age had no effect on ERalpha or ERbeta expression in non-HT or premenopausal women. CONCLUSIONS: ERbeta is the predominant ER in human coronary arteries and correlates with coronary calcification, a marker of severe atherosclerosis. Increased ERbeta expression is linked to advanced atherosclerosis and calcification independent of age or hormone status. Future pharmacogenetic studies that target this receptor are needed to confirm causality.

Interobserver reproducibility of criteria for vertebral body exclusion.

UNLABELLED: We studied reproducibility of the ISCD vertebral exclusion criteria among four interpreters. Surprisingly, agreement among interpreters was only moderate, because of differences in threshold for diagnosing focal structural defects and choice of which vertebra among a pair discordant for T-score, area, or BMC to exclude. Our results suggest that reproducibility may be improved by specifically addressing the sources of interobserver disagreement. INTRODUCTION: Although DXA is widely used to measure vertebral BMD, its interpretation is subject to multiple confounders including osteoarthritis, aortic calcification, and scoliosis. In an attempt to standardize interpretation and minimize the impact of artifacts, the International Society for Clinical Densitometry (ISCD) established criteria for vertebral exclusion, including the presence of a focal structural defect (FSD), discrepancy of >1 SD in T-score between adjacent vertebrae, and a lack of increase in BMC or area from L1 to L4. Whereas the efforts of the ISCD represent an important advance in BMD interpretation, the interobserver reproducibility with application of these criteria is unknown. We hypothesized that there would be substantial agreement among four interpreters regarding application of the exclusion criteria and the final lumbar spine T-score. MATERIALS AND METHODS: Each interpreter read a set of 200 lumbar DXA scans obtained on male veterans, applying the ISCD vertebral body exclusion criteria. RESULTS: Surprisingly, agreement among interpreters was only moderate. Differences in interpretation resulted from differing thresholds for recognition of FSD and the choice of excluding the upper or lower vertebral body for the criteria requiring comparison between adjacent vertebrae. CONCLUSIONS: Despite their apparent simplicity, the ISCD vertebral exclusion criteria are difficult to apply consistently. In principle, appropriate refinement of the exclusion criteria may significantly improve interobserver agreement.

Correlating androgen and estrogen steroid receptor expression with coronary calcification and atherosclerosis in men without known coronary artery disease.

BACKGROUND: Accumulating data emphasize the gender specificity of key components of the atherosclerotic process and the importance of gonadal steroids on the human vasculature. Steroid receptors, including the androgen receptor (AR) and estrogen receptors (ERs) alpha and beta are expressed in key vascular tissues, including endothelial cells and vascular smooth muscle cells. However, the relative abundance and importance of these receptors in the coronary artery are not well defined, particularly in men. We therefore examined AR, ER alpha, and ER beta expression as a function of key components of atherosclerosis, namely plaque and calcium area, in male human coronary arteries. METHODS: Coronary arteries were obtained at autopsy from 24 men without known coronary artery disease. Coronary calcification was measured by contact microradiography, and atherosclerotic plaque area was quantified histologically. Coronary artery cross-sections were immunostained for AR, ER alpha, and ER beta and then measured semiquantitatively in each arterial wall layer (intima, adventitia, and media). RESULTS: AR, ER beta, and ER alpha were expressed in all artery wall layers but most avidly in the media (P < 0.001). ER beta exceeded ER alpha expression (P < 0.0005). AR expression in the media correlated negatively with plaque area (P = 0.006, R = -0.55), whereas intimal ER beta expression correlated positively with plaque area (P = 0.012, R = 0.50). CONCLUSIONS: We conclude that both AR and ER beta are important in relatively early coronary atherosclerosis, but inversely so, because decreasing AR and increasing ER beta expression correlate with more extensive atherosclerosis. ER beta seems to be the predominate ER in coronary arteries harvested from men without known coronary artery disease. Interventional studies are required to assess the functional significance of these observations.

Prevalence and predictors of coronary artery calcification in women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS), a common endocrine disorder of reproductive-aged women, is associated with multiple risk factors for coronary heart disease (CHD), such as diabetes mellitus, dyslipidemia, visceral obesity, and hypertension. However, premature coronary atherosclerosis has not been demonstrated in PCOS women. Electron beam computed tomography (EBCT) noninvasively measures coronary artery calcium (CAC), a marker for coronary atherosclerosis. We measured CAC by EBCT in 30- to 45-yr-old premenopausal PCOS women and compared the results to CAC in 1) recruited normal ovulatory volunteers matched for age and weight to the PCOS cohort, and 2) community-dwelling women of similar age in an extant coronary calcium database. Healthy, community-dwelling, ovulatory controls (n = 71) were matched by age and body mass index (BMI) to PCOS women (n = 36). Women with diabetes or known CHD were excluded. Subjects underwent EBCT scanning, oral glucose tolerance testing, and CHD risk factor assessment. PCOS women had significantly higher levels of serum total and low density lipoprotein cholesterol and testosterone levels than matched controls. PCOS and control women were obese and had a greater mean BMI than community-dwelling women (33 kg/m(2) for PCOS vs. 31 kg/m(2) for control; P < 0.001). CAC was more prevalent in PCOS women (39%) than in matched controls (21%; odds ratio, 2.4; P = 0.05) or community-dwelling women (9.9%; odds ratio, 5.9; P < 0.001). BMI, waist circumference, and total and low density lipoprotein cholesterol levels predicted CAC prevalence after adjustment for BMI. CAC is more prevalent in PCOS women than in obese or nonobese women of similar age. PCOS women are at increased risk for atherosclerosis and should be targeted for primary prevention of CHD.