Elevated Serum Markers Research Articles

Direct-Acting Antiviral Treatment of Patients with Hepatitis C Resolves Serologic and Histopathologic Features of Autoimmune Hepatitis.

Patients with hepatitis C virus (HCV) often have elevated serum markers and histologic features of autoimmune hepatitis (AIH). We evaluated an HCV‐positive (HCV+) study group that had elevated serum markers of AIH before starting direct‐acting antiviral (DAA) therapy (n = 21) and compared them to an HCV+ control group that did not have laboratory studies suggesting AIH (n = 21). Several patients in the study (17/21) and control (11/21) groups had liver biopsies before DAA treatment, and many were biopsied due to elevated serum markers of AIH. Evaluation of pre‐DAA treatment liver biopsies showed histologic features suggestive of AIH in 64.7% (11/17) of the study group and 45.5% (5/11) of the control group. Patients who were HCV+ with elevated serum markers of AIH had significantly increased hepatitis activity (P < 0.001) and slightly increased fibrosis stages (P = 0.039) in their pretreatment liver biopsies compared to controls. We hypothesized that the elevated serum markers and histologic features of AIH would resolve following DAA treatment. Serum markers of AIH in the study group began decreasing by 6 months posttreatment, and 52.4% (11/21) had complete resolution. Alanine aminotransferase levels significantly decreased into the normal range for all patients (21/21). Even patients that had persistence of serum markers of AIH after DAA treatment had normal transaminases. Six patients from the study patient group and 4 patients from the control group had follow‐up liver biopsies after DAA treatment, and all biopsies showed resolution of the histologic features of AIH. Conclusion: The majority of HCV+ patients that have serum markers and/or histopathologic features of AIH should initially be treated with DAA.

Elevated Serum Markers of Acute Kidney Injury in Patients With Obstructive Sleep Apnea.

Previous research revealed a positive correlation between endothelial cell injury (indicated by albuminuria) and obstructive sleep apnea (OSA). However, little else has been revealed about acute kidney injury (AKI) in patients with OSA. This prospective study recruited consecutive patients undergoing overnight polysomnography for evaluation of sleep apnea. Patients in whom any major disease or recent infection had been previously diagnosed were excluded. Ultimately, data from 75 patients with apnea-hypopnea indices of 5 or more were analyzed. Baseline values for the urinary albumin-creatinine ratio (UACR), serum levels for three markers of AKI (cystatin C, neutrophil gelatinase-associated lipocalin [NGAL], interleukin-18 [IL-18]), and polysomnography data were recorded and analyzed. Patients then were followed for 6 months of continuous positive airway pressure (CPAP) treatment. At baseline, UACRs were greater in patients with more severe OSA (P = .005, r = .329). All three serum markers of AKI (cystatin C, NGAL, and IL-18) studied were positively correlated with OSA severity, and two (cystatin C and IL-18) were positively correlated with the frequency of oxygen desaturation during sleep. However, none of the AKI markers had positive correlations with UACR. After 6 months of CPAP treatment, UACR and IL-18 were decreased significantly in patients with good adherence. Albuminuria and levels of three serum markers of AKI (cystatin C, NGAL, IL-18) were positively correlated with OSA severity, and good adherence with CPAP treatment decreased albuminuria and interleukin-18 levels. These results may provide additional tools for assessing early renal injury in patients with OSA.

Possible Hepatoprotective Effects of Mustard Seed Extract Against Paracetamol-Induced Liver Injury in Male Albino Rat

The evaluation of hepatoprotective effect of Mustard seed (MS) extract was assessed against paracetamol induced hepatic injury in albino rats. Animals were divided into 4 groups, each of 5 rats. Group I served as control group; group II treated with ethanolic extract from Mustard seed at a dose level of 500 mg/kg body weight orally once for 10 days. Group III represents induced liver injury animals using single dose of paracetamol (PCT). Group IV combined treatment of MS extract and PCT. Elevated serum marker enzymes such as ALT, AST, ALP, GGT and LDH were observed due to paracetamol treatment. Combined administration of MS extract with PCT to rat partially normalized the altered hepatic biochemical markers caused by PCT. Moreover, PCT caused histological changes in livers of rat including swelling and dilation in sinusoidal space, inflammation in portal tract and hepatocytes, as well as, hyperplasia in Kuppfer cells. However, co-administration of MS extract with PCT alleviated to some extent the changes caused by PCT but not as the normal liver. The results of this study strongly indicate that Mustard seed has possible potent hepatoprotective action against paracetamol induced hepatic damage in rats.

Possible Hepatoprotective Effects of Mustard Seed Extract Against Paracetamol-Induced Liver Injury in Male Albino Rat

The evaluation of hepatoprotective effect of Mustard seed (MS) extract was assessed against paracetamol induced hepatic injury in albino rats. Animals were divided into 4 groups, each of 5 rats. Group I served as control group; group II treated with ethanolic extract from Mustard seed at a dose level of 500 mg/kg body weight orally once for 10 days. Group III represents induced liver injury animals using single dose of paracetamol (PCT). Group IV combined treatment of MS extract and PCT. Elevated serum marker enzymes such as ALT, AST, ALP, GGT and LDH were observed due to paracetamol treatment. Combined administration of MS extract with PCT to rat partially normalized the altered hepatic biochemical markers caused by PCT. Moreover, PCT caused histological changes in livers of rat including swelling and dilation in sinusoidal space, inflammation in portal tract and hepatocytes, as well as, hyperplasia in Kuppfer cells. However, co-administration of MS extract with PCT alleviated to some extent the changes caused by PCT but not as the normal liver. The results of this study strongly indicate that Mustard seed has possible potent hepatoprotective action against paracetamol induced hepatic damage in rats.

The Clinical Significance of a Small Component of Choriocarcinoma in Testicular Mixed Germ Cell Tumor (MGCT).

The clinical significance of limited choriocarcinoma in a malignant mixed germ cell tumor (MGCT) is unknown. Men with a MGCT with ≤5% choriocarcinoma at radical orchiectomy (RO) between 2000 and 2016 from our consult service were studied. Of 50 men in our cohort, we had clinical information for 30 men. Median follow-up was 41 months (1 to 168 mo). Median tumor size was 4.5 cm (1.1 to 8.0 cm). In total, 22/30 (73%) cases were pT1, 6/30(20%) cases were pT2, and 2/30 (7%) cases were pT3. In total, 4/30(13%) cases had lymph node metastases and 2/30 (7%) cases had distant metastases at the time of RO. In 30 cases with RO we had information on immediate postorchiectomy treatment: 14/30 (46.7%) active surveillance, 4/30 (13.3%) retroperitoneal lymph node dissection, 10/30 (33.3%) chemotherapy (chemotherapy), 1/30 (3.3%) retroperitoneal lymph node dissection followed by chemotherapy, and 1/30 (3.3%) resection of a distant metastasis. Preoperative serum human chorionic gonadotropin (hCG) levels ranged between 0.1 and 60,715 mIU/mL (mean, 4796; median, 485). One patient had an hCG level of 6367 mIU/mL and another 60,715 mIU/mL with the remaining cases <5000 mIU/mL. In total, 4/30 (13%) patients had elevated serum markers after surgery, 3 of them normalized following chemotherapy while the fourth one continued to have elevated serum alpha fetoprotein levels after chemotherapy. All patients were alive at last follow-up. In total, 7/30 (23.3%) patients subsequently developed metastatic disease to lymph nodes or distal organs, the histology of the metastasis consisted mainly of teratoma and yolk sac tumor. Embryonal carcinoma was present in 2 metastatic sites. One lung metastasis was suggestive for choriocarcinoma. Definitive choriocarcinoma was not present in any of the metastasis. A small component of choriocarcinoma in a MGCT is typically associated with relatively low-level elevations of serum hCG levels, and is not associated with aggressive disease. The presence of limited choriocarcinoma (≤5%) does not add to the prognostic information provided by standard TNM staging, which uses levels of serum markers (hCG, alpha fetoprotein, lactate dehydrogenase) as surrogates for extent of disease.

An ellagic acid isolated from Clerodendrum viscosum leaves ameliorates iron-overload induced hepatotoxicity in Swiss albino mice through inhibition of oxidative stress and the apoptotic pathway

Iron is a vital element required for normal cellular physiology in animal systems, but excess iron accumulation in the biological system accelerates oxidative stress, cellular toxicity, tissue injury and organ fibrosis, which ultimately leads to the generation of chronic liver diseases including cancer. A natural antioxidant, ellagic acid (EA) has been previously reported for its pharmacological properties; however, there is no significant evidence available that could illustrate its protective potential against iron-overload induced hepatotoxicity. In the present work, EA was evaluated for its in vitro free radical scavenging and iron chelation potentials. Further, EA was tested in vivo for its protective activity against iron overload-induced hepatotoxicity in Swiss albino mice by evaluating liver iron content, reactive oxygen species (ROS), liver antioxidant enzymes, serum marker levels, liver damage and fibrosis, histopathological study and finally western blotting analysis. EA treatment significantly decreased liver iron and serum ferritin levels. Elevated ROS levels, decreased antioxidant parameters and elevated serum markers were normalized upon treatment with EA. Cellular morphology, iron -overload and liver fibrosis were found to be effectively ameliorated. Finally, the protective effect of EA against iron overload-induced apoptosis was confirmed by western blotting when its treatment upregulated the expressions of caspase-3 and poly(ADP-ribose) polymerase (PARP) proteins. EA revealed hepatoprotective activity against iron overload-induced toxicity through scavenging free radicals, inhibiting excess ROS production, normalizing liver damage parameters and upregulating caspase-3, PARP expression. Collectively, our findings support the possible use of the natural antioxidant EA as a promising candidate against iron-overloaded diseases.

COMPARATIVE STUDY OF BUTANOLIC AND ETHANOLIC EXTRACTS OF TRIBULUS TERRESTRIS FRUITS IN 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN-INDUCED HEPATOTOXICITY IN RATS

Objective: To compare hepatoprotective activity of ethanol and butanol fraction of Tribulus terrestris fruits against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hepatic damage in albino rats.Methods: The degree of liver protection was assessed in terms of biochemical parameters, liver marker enzymes, and lipid peroxidation (LPO) in drug-treated and TCDD-intoxicated rats.Results: The results revealed that the elevated serum marker enzymes and biochemical parameters in TCDD-treated rats were significantly reduced toward normal levels in saponin rich fraction of T. terrestris drug-treated animals when comparable to ethanolic extract of T. terrestris drug-treated group. The plant extracts have got a notable inhibitory activity on the formation of LPO products in the basal as well as in the presence of inducers.Conclusion: The plant extracts hindered oxidative damage that occurred to the hepatocytes during TCDD toxicity may be responsible for the hepatoprotective activity in the present study.

Eosinophilic Esophagitis: an Important Comorbid Condition of Asthma?

Eosinophilic esophagitis and asthma are frequently found as comorbid conditions in children and adults along with other manifestations of atopic diathesis. These two conditions have similar T helper 2 responses-driven pathophysiology and share common management strategies such as using systemic corticosteroids and targeted anti-cytokine biologic therapies. Review of the literature finds that asthma is often a comorbid condition in eosinophilic esophagitis in both children and adults; however, the EoE-asthma relationship remains poorly characterized mechanistically and clinically. EoE and asthma commonly share several comorbid conditions such as allergic rhinitis and gastroesophageal reflux disease; therefore, addressing these comorbid conditions has the potential to improve and/or maintain control in both diseases. Similar to asthma, patients with EoE frequently demonstrate elevations in serum markers of atopy, including serum IgE levels, peripheral eosinophil counts, and T helper 2-related cytokines. Gastroesophageal reflux disease is thought to affect asthma through microaspirations, airway hyperresponsiveness, and increased vagal tone. The understanding of the relationship between gastroesophageal reflux and EoE is still evolving but seems to be bidirectional and interactive. In terms of treatment, similar classes of medications have been used in both EoE and asthma. In both children and adults, EoE remission can be achieved by food trigger avoidance and use of corticosteroids and biologic therapies. Asthma control is mostly achieved through inhaled corticosteroids, and long but biologic therapies are increasingly used in severe subsets of the disease. Significant clinical and mechanistic work needs to be accomplished to better understand the relationship between asthma, EoE, and their interaction with other allergic diseases. Understanding whether shared mechanisms exist can lead to the development of new diagnostic and therapeutic strategies. The following review examines the existing literature regarding prevalence, common comorbidities, and potential therapeutic approach and identifies gaps in knowledge and future directions.

Baseline IL-2 and the AIH score can predict the response to standard therapy in paediatric autoimmune hepatitis

Although autoimmune hepatitis (AIH) can be treated with corticosteroid-based first-line therapy, incomplete remission is associated with progressive liver fibrosis. So far accepted predictors of the subsequent treatment response of AIH patients are lacking. Therefore, we analysed baseline parameters, including iron homeostasis and cytokine levels, in 60 children with paediatric AIH (pAIH). In contrast to adults, elevated serum markers indicating iron overload were not commonly found in children. Therefore, ferritin was not predictive of the treatment response in pAIH. Although baseline immunoglobulins were lower in pAIH children with subsequent complete biochemical remission (BR) upon standard first-line therapy, only lower AIH scores (≤16 points) could predict BR upon standard therapy in our training and validation cohorts. Additionally, higher baseline IL-2 and MCP-1/CCL2 levels were associated with BR in a sub-cohort. A combined score of IL-2 level and a simplified AIH score predicted treatment response more precisely than both parameter alone in this sub-cohort. In conclusion, the baseline AIH score could be validated as a predictor of treatment response in pAIH. Additionally, low baseline IL-2 may help identify children who need salvage therapy. This could be important because the use of low-dose IL-2 therapies is being tested in various autoimmune diseases.

Novel Diagnostic and Predictive Biomarkers in Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease for a multitude of reasons including very late diagnosis. This in part is due to the lack of understanding of the biological behavior of PDAC and the ineffective screening for this disease. Significant efforts have been dedicated to finding the appropriate serum and imaging biomarkers to help early detection and predict response to treatment of PDAC. Carbohydrate antigen 19-9 (CA 19-9) has been the most validated serum marker and has the highest positive predictive value as a stand-alone marker. When combined with carcinoembryonic antigen (CEA) and carbohydrate antigen 125 (CA 125), CA 19-9 can help predict the outcome of patients to surgery and chemotherapy. A slew of novel serum markers including multimarker panels as well as genetic and epigenetic materials have potential for early detection of pancreatic cancer, although these remain to be validated in larger trials. Imaging studies may not correlate with elevated serum markers. Critical features for determining PDAC include the presence of a mass, dilated pancreatic duct, and a duct cut-off sign. Features that are indicative of early metastasis includes neurovascular bundle involvement, duodenal invasion, and greater post contrast enhancement. 18-F-fluorodeoxyglucose (18-FDG) radiotracer uptake and changes following treatment may predict patient overall survival following treatment. Similarly, pretreatment apparent diffusion coefficient (ADC) values may predict prognosis with lower ADC lesions having worse outcome. Although these markers have provided significant improvement in the care of pancreatic cancer patients, further advancements can be made with perhaps better combination of markers or discovery of unique marker(s) to pancreatic cancer.

Patterns of relapse and second malignancy after adjuvant external beam radiation therapy for stage I-II seminoma.

428 Background: To determine relapse rate, patterns of relapse and risk of second malignancy (SM) from a large single institution experience of adjuvant external beam radiation therapy (RT) for stage I-II seminoma. Methods: 463 patients with clinical stage I (n = 339) and II (n = 124) seminoma underwent adjuvant RT (median 25.5Gy) between 2/1990 and 11/2015. Data was gathered by retrospective chart review. Patients with &gt; 5 years of follow-up (n = 312) were included in analysis of SM risk. Of stage II patients, 72% (89/124) received a boost to gross disease (median 4.5Gy). Field design was para-aortic nodal region only (n = 96), para-aortic and ipsilateral pelvic nodal region (n = 351) or para-aortic and bilateral pelvic nodal region (n = 8). Field design was not available for 8 patients. Patients were followed with clinical exam, serial imaging, and tumor markers. Relapse and SM were confirmed pathologically. Results: At median follow-up of 7.9 years, there were 20 relapses (median 13.2 months; range 2.5-55.3 months). There were 9 and 11 relapses in stage I and II patients, respectively, with 7/20 (35%) occurring &gt; 2 years after RT. Relapses were identified by clinical symptoms (n = 7), imaging (n = 9), or elevated serum markers (n = 4). Sites of relapses included the lung/mediastinum (n = 10), retroperitoneum/pelvis (n = 5), bone (n = 3) and inguinal nodes (n = 2). 15 (3 pelvic) occurred after para-aortic and ipsilateral pelvic lymph node RT, while 5 (2 pelvic) occurred after para-aortic RT alone. 19/20 patients received cisplatin-based chemotherapy for relapse and were without evidence of disease at last follow-up (median 123 months). Of 35 total non-testicular SM (33 patients), 17 (48.6%) were in the RT field, 4 (11.4%) were marginal and 14 (40%) were out of field. Common SM were prostate (10), lymphoma (4), bladder (3) and kidney (3). The 5 and 10 year overall survival for the cohort is 99.2% and 97.9%, respectively. Conclusions: Stage I-II seminoma patients have a low risk of relapse and SM following adjuvant RT. Relapse is less common in patients treated with para-aortic and pelvic fields. More than 1/3 of relapses occur more than 2 years after adjuvant RT, necessitating long term clinical, radiographic and biochemical follow-up.

A 35 kDa Phyllanthus niruri protein suppresses indomethacin mediated hepatic impairments: Its role in Hsp70, HO-1, JNKs and Ca2+ dependent inflammatory pathways

The present study has been conducted to explore a novel strategy to modulate the unfavourable effects of indomethacin by Phyllanthus niruri protein (PNP) and the underlying mechanism PNP exploits for the amelioration of that pathophysiology. In hepatocytes, indomethacin enhanced reactive oxygen species (ROS), reduced intracellular antioxidant capacity, up regulated mitogen activated protein kinase (MAPKs), disrupted mitochondrial membrane potential, activated apoptotic pathways and there by reduced the viability of the hepatocytes. Simultaneous treatment with PNP modulated these detrimental actions of the drug and retained cell viability. Similarly, in mice, indomethacin elevated serum marker enzymes (e.g. Alanine Transaminase), decreased antioxidant enzyme activities, elevated oxidations of lipids and proteins, increased intracellular calcium overload mediated endoplasmic reticular stress (ER stress) pathways, up regulated the pro-inflammatory cytokines and there by leading to the mitochondrial dependent caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. Moreover investigation of these inherent molecular pathways exhibited that these alterations are associated with up regulation of MAPKs, inducible nitric oxide synthase (iNOS), heme oxygenase-1 and down regulation of survival proteins. However, PNP suppressed those apoptotic indices as evidenced from histopathological studies and DNA fragmentation analysis. Combining, results suggest that PNP could possibly provide a protection against indomethacin-induced hepatic pathophysiology.

Intestinal dysbiosis is common in systemic sclerosis and associated with gastrointestinal and extraintestinal features of disease.

BackgroundRecent evidence suggests a link between autoimmunity and the intestinal microbial composition in several rheumatic diseases including systemic sclerosis (SSc). The objective of this study was to investigate the prevalence of intestinal dysbiosis in SSc and to characterise patients suffering from this potentially immunomodulatory deviation.MethodsThis study consisted of 98 consecutive patients subject to in-hospital care. Stool samples were analysed for intestinal microbiota composition using a validated genome-based microbiota test (GA-map™ Dysbiosis Test, Genetic Analysis, Oslo, Norway). Gut microbiota dysbiosis was found present as per this standardised test. Patients were examined regarding gastrointestinal and extraintestinal manifestations of SSc by clinical, laboratory, and radiological measures including esophageal cineradiography, the Malnutrition Universal Screening Tool (MUST), levels of plasma transthyretin (a marker of malnutrition) and faecal (F-) calprotectin (a marker of intestinal inflammation).ResultsA majority (75.5%) of the patients exhibited dysbiosis. Dysbiosis was more severe (rs = 0.31, p = 0.001) and more common (p = 0.013) in patients with esophageal dysmotility. Dysbiosis was also more pronounced in patients with abnormal plasma levels of transthyretin (p = 0.045) or micronutrient deficiency (p = 0.009). In 19 patients at risk for malnutrition according to the MUST, 18 exhibited dysbiosis. Conversely, of the 24 patients with a negative dysbiosis test, only one was at risk for malnutrition. The mean ± SEM levels of F-calprotectin were 112 ± 14 and 45 ± 8 μg/g in patients with a positive and negative dysbiosis test, respectively. Dysbiosis was more severe in patients with skin telangiectasias (p = 0.020), pitting scars (p = 0.023), pulmonary fibrosis (p = 0.009), and elevated serum markers of inflammation (p < 0.001). However, dysbiosis did not correlate with age, disease duration, disease subtype, or extent of skin fibrosis.ConclusionsIn this cross-sectional study, intestinal dysbiosis was common in patients with SSc and was associated with gastrointestinal dysfunction, malnutrition and with some inflammatory, fibrotic and vascular extraintestinal features of SSc. Further studies are needed to elucidate the potential causal relationship of intestinal microbe-host interaction in this autoimmune disease.

A Novel ANO5 Mutation Causing Gnathodiaphyseal Dysplasia With High Bone Turnover Osteosclerosis.

Gnathodiaphyseal dysplasia (GDD) is a rare skeletal syndrome that involves an osteopetrosis-like sclerosis of the long bones and fibrous dysplasia-like cemento-osseous lesions of the jawbone. Although the genetic analysis of the respective patients has revealed mutations in the ANO5 gene as an underlying cause, there is still no established consensus regarding the bone status of GDD patients. We report a new case of GDD in a 13-year-old boy with recurrent diaphyseal fractures of the femur, in whom we identified a novel de novo missense mutation in the ANO5 gene, causing a p.Ser500Phe substitution at the protein level. After confirming the presence of GDD-characteristic abnormalities within the jaw bones, we focused on a full osteologic assessment using dual-energy X-ray absorptiometry (DXA), high-resolution peripheral quantitative computed tomography (HR-pQCT), and serum analyses. We thereby identified increased trabecular bone mass accompanied by elevated serum markers of bone formation and bone resorption. The high turnover bone pathology was further confirmed through the analysis of an iliac crest biopsy, where osteoblast and osteoclast indices were remarkably increased. Taken together, our findings provide evidence for a critical and generalized role of anoctamin-5 (the protein encoded by the ANO5 gene) in skeletal biology. As it is reasonable to speculate that modifying the function of anoctamin-5 might be useful for therapeutically activating bone remodeling, it is now required to analyze its function at a molecular level, for instance in mouse models. © 2016 American Society for Bone and Mineral Research.

Chronic oral administration of low-dose combination of fenofibrate and rosuvastatin protects the rat heart against experimentally induced acute myocardial infarction.

Fenofibrate and rosuvastatin at low doses might have experimental pleiotropic benefits. This study investigated the combined effect of low doses of fenofibrate and rosuvastatin in isoproterenol-induced experimental myocardial infarction. Rats administered isoproterenol (85 mg/kg/day, s.c.) for 2 days (day 29 and day 30) of 30 days experimental protocol developed significant myocardial infarction that was accompanied with high myocardial oxidative stress and lipid peroxidation, elevated serum markers of cardiac injury, lipid abnormalities, and elevated circulatory levels of C-reactive protein. Pretreatment with low doses of fenofibrate (30 mg/kg/day p.o., 30 days) and rosuvastatin (2 mg/kg/day p.o., 30 days) both alone or in combination markedly prevented isoproterenol-induced myocardial infarction and associated abnormalities while the low-dose combination of fenofibrate and rosuvastatin was more effective. Histopathological study in isoproterenol control rat heart showed necrosis with edema and acute inflammation at the margins of necrotic area. The rat heart from low-dose fenofibrate and rosuvastatin pretreated group showed scanty inflammation and no ischemia. In conclusion, fenofibrate and rosuvastatin pretreatment in low doses might have a therapeutic potential to prevent the pathogenesis of myocardial infarction. Moreover, their combined treatment option might offer superior therapeutic benefits via a marked reduction in myocardial infarct size and oxidative stress, suggesting a possibility of their pleiotropic cardioprotective action at low doses.

Chromogranin A and neurone-specific enolase serum levels as predictors of treatment outcome in patients with metastatic castration-resistant prostate cancer undergoing abiraterone therapy.

To determine the impact of elevated neuroendocrine serum markers on treatment outcome in patients with metastatic castration-resistant prostate cancer (mCRPC) undergoing treatment with abiraterone in a post-chemotherapy setting. Chromogranin A (CGa) and neurone-specific enolase (NSE) were determined in serum drawn before treatment with abiraterone from 45 patients with mCRPC. Outcome measures were overall survival (OS), prostate-specific antigen (PSA) response defined by a PSA level decline of ≥50%, PSA progression-free survival (PSA-PFS), and clinical or radiographic PFS. The CGa and NSE serum levels did not correlate (P = 0.6). Patients were stratified in to low- (nine patients), intermediate- (18) or high-risk (18) groups according to elevation of none, one, or both neuroendocrine markers, respectively. The risk groups correlated with decreasing median OS (median OS not reached vs 15.3 vs 6.6 months; P < 0.001), decreasing median clinical or radiographic PFS (8.3 vs 4.4 vs 2.7 months; P = 0.001) and decreasing median PSA-PFS (12.0 vs 3.2 vs 2.7 months; P = 0.012). In multivariate Cox regression analysis the combination of CGa and NSE (≥1 marker positive vs both markers negative) remained significant predictors of OS, clinical or radiographic PFS, and PSA-PFS. We did not observe a correlation with PSA response (63% vs 35% vs 31%; P = 0.2). Chromogranin A and NSE did not predict PSA response in patients with mCRPC treated with abiraterone. However, we observed a correlation with shorter PSA-PFS, clinical or radiographic PFS, and OS. This might be due to an elevated risk of developing resistance under abiraterone treatment related to neuroendocrine differentiation.

Abstract B10: FOXL2 402C&amp;gt;G mutation can be identified in the circulating tumor DNA of patients with adult-type granulosa cell tumor.

Abstract Background: Adult granulosa cell tumors of the ovary are molecularly unique as 94% of tumours are defined by the pathognomonic FOXL2 402C&amp;gt;G C134W mutation. More accurate methods are needed for the diagnosis and follow-up of AGCT patients. This study was designed to evaluate circulating tumor DNA (ctDNA) FOXL2 mutation as a diagnostic and monitoring tool in AGCT patients. Methods: An optimized ddPCR assay was performed on free ctDNA extracted from 120 serial plasma samples (1-2mL) collected prospectively from 35 AGCT patients during 8/2007-4/2014 in Helsinki, Finland and in Vancouver, Canada. Clinical follow-up information was retrieved from hospital records. The median follow-up time was 6.3 (range 1.1 – 33.9) years. The statistical analyses were performed with JMP Pro 10 software. Results: Of the 35 AGCT patients, 33 (31 Finland, 2 Vancouver) patients had measurable disease at the time of sample collection, and 12 (36%) harbored a detectable plasma ctDNA FOXL2 mutation. The FOXL2 mutation was identified in the ctDNA of 6/17 (36.3%) patients with primary, and 6/31 (19.4%) of patients with recurrent disease, respectively. Median tumor size was significantly larger in the mutation positive cases (13.5cm) compared to the mutation negatives (7.5cm; p=0.003), and the mutation was present in 9/16 (56%) of samples from patients with tumors over 10cm in diameter. Similarly, ctDNA FOXL2 mutation frequency positively correlated to tumor size (Spearman's Rho=0.32, p=0.003). The sensitivity of the ddPCR assay to detect AGCT disease presence was 25%, while the specificity was 90% (ROC-AUC 0.58). In patients with primary tumors, the ctDNA FOXL2 positivity did not correlate with tumor stage, and none of these patients has presented with a tumor recurrence during follow-up. This is putatively due to the relatively short follow-up time of (3.6 years, range 1.1-6.3 years). Given that the median time to relapse has been reported to be 7-8 years, no definitive conclusions can be drawn on the effect of ctDNA FOXL2 on the risk of AGCT recurrence. We identified the presence of low-level ctDNA FOXL2 mutations in four patient's plasma that did not have clinical disease present. From patient ID 208, the first sample was drawn 7 months post primary tumor operation and already then the FOXL2 mutation was detected at 1.4% frequency. Subsequently, the FOXL2 ctDNA mutation was detected at two time points during follow-up, before the elevation of serum markers and clinical relapse with multiple metastases. From patient ID 221, we identified the ctDNA FOXL2 mutation in a plasma sample taken upon primary surgery. However, the FOXL2 mutation was identified in plasma samples taken at 10 and 34 months post primary surgery. This patient was last monitored with no clinical signs of relapse 49 months post surgery. In two patients (ID 213, 216), the FOXL2 mutation was detected at primary diagnosis, then once (at 24 months for ID 213, and at 38 months for ID 216) post primary surgery, and no subsequent relapse had been detected for these two patients during follow-up. In total, we identified three patients with the ctDNA FOXL2 mutation present in the plasma with no evidence of subsequent recurrence. Since the FOXL2 mutation is specific to AGCT, the only source of ctDNA FOXL2 mutation is likely to be from occult AGCT cells, and further monitoring of these patients over time will reveal if they will develop a clinical AGCT recurrence. Conclusions: As a proof of concept, we established that specific molecular diagnosis and detection of primary and recurrent AGCT can be achieved non-invasively with a “liquid biopsy” and ctDNA FOXL2 mutation testing. Further studies are needed to increase the sensitivity and clinical applicability of ctDNA FOXL2 mutation testing in AGCT patients. Citation Format: Anniina Färkkilä, Melissa K. McConechy, Winnie Yang, Aline Talhouk, Ying Ng, Ryan Morin, Annika Riska, Jessica N. McAlpine, Leila Unkila-Kallio, Mikko Anttonen, David G. Huntsman. FOXL2 402C&amp;gt;G mutation can be identified in the circulating tumor DNA of patients with adult-type granulosa cell tumor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B10.

The NAE inhibitor pevonedistat (MLN4924) synergizes with TNF-α to activate apoptosis

Predicting and understanding the mechanism of drug-induced toxicity is one of the primary goals of drug development. It has been hypothesized that inflammation may have a synergistic role in this process. Cell-based models provide an easily manipulated system to investigate this type of drug toxicity. Several groups have attempted to reproduce in vivo toxicity with combination treatment of pharmacological agents and inflammatory cytokines. Through this approach, synergistic cytotoxicity between the investigational agent pevonedistat (MLN4924) and TNF-α was identified. Pevonedistat is an inhibitor of the NEDD8-activating enzyme (NAE). Inhibition of NAE prevents activation of cullin-RING ligases, which are critical for proteasome-mediated protein degradation. TNF-α is a cytokine that is involved in inflammatory responses and cell death, among other biological functions. Treatment of cultured cells with the combination of pevonedistat and TNF-α, but not as single agents, resulted in rapid cell death. This cell death was determined to be mediated by caspase-8. Interestingly, the combination treatment of pevonedistat and TNF-α also caused an accumulation of the p10 protease subunit of caspase-8 that was not observed with cytotoxic doses of TNF-α. Under conditions where apoptosis was blocked, the mechanism of death switched to necroptosis. Trimerized MLKL was verified as a biomarker of necroptotic cell death. The synergistic toxicity of pevonedistat and elevated TNF-α was also demonstrated by in vivo rat studies. Only the combination treatment resulted in elevated serum markers of liver damage and single-cell hepatocyte necrosis. Taken together, the results of this work have characterized a novel synergistic toxicity driven by pevonedistat and TNF-α.

Sa1681 Mitochondria-Targeting Antioxidant Attenuates Cholestasis-Induced Liver Fibrosis in Mice

BACKGROUND: Hepatic fibrosis is seen in patients with chronic liver injury. It occurs when damaged regions of the liver undergo unregulated production and degradation of the extracellular matrix. Hepatic fibrosis pathology is frequently reported in 5 stages (0,1,2,3,4): stage 0 representing no fibrosis and stage 4 representing advanced bridging fibrosis with cirrhosis. The gold standard for establishing the stage of fibrosis has been a liver biopsy. It is a preconceived notion that transaminase markers remain normal across the stages of fibrosis, however there has been limited published data to support this claim. We set out to prove: (a) serum markers remain normal amongst stages of hepatic fibrosis and (b) lack of correlation between lab values and progressive stages of fibrosis. METHODS: A retrospective observational study was conducted evaluating liver biopsies from 2010 to 2014. Serum markers (AST, ALT, INR, BUN) were extracted within 90 days before and after liver biopsy. Acute decompensated liver failure patients were also excluded. Mean and 95% confidence intervals (95%CI) were used to describe the distributions of serum markers in different fibrosis stages. A logistic regression model was used to estimate the relative risk of elevated serum markers (over normal value) in the fibrosis stages 1-4 compared to in the stage 0. P-values and 95 percent confidence intervals were calculated using the logistic regression model. All analyses were done in SAS 9.4 (SAS Institute Inc., Cary, NC, USA). P<0.05 was considered as statistically significant. RESULTS: A total of 771 liver biopsies were screened, after exclusion criteria were applied, roughly 600 samples were analyzed. AST 95% confidence interval for fibrosis stage 0 overlapped with all stages of fibrosis including stage 4. ALT 95% confidence interval of fibrosis stage 0, 1, and 4 overlapped. BUN 95% confidence interval for fibrosis stage 0 overlapped with all stages of fibrosis including stage 4. INR 95% confidence interval for fibrosis stage 0 overlapped with all stages of fibrosis, except stage 4. Conclusion: Lab values (AST, ALT) are poor predictors of chronic liver disease. Specifically, these lab values may remain statistically the same throughout the spectrum of chronic liver disease ranging from (stage 0) to cirrhosis (stage 4). One statistically significant value was represented by INR in stage 4 fibrosis, consistent with diminished synthetic function amongst these patients. With the rapidly rising incidence of chronic liver disease largely in part due to NAFLD, it is imperative for clinicians to keep in mind: normal serum markers don't convey a disease free liver.

Growth hormone resistance exacerbates cholestasis-induced murine liver fibrosis.

Growth hormone (GH) resistance has been associated with liver cirrhosis in humans but its contribution to the disease remains controversial. In order to elucidate whether GH resistance plays a causal role in the establishment and development of liver fibrosis, or rather represents a major consequence thereof, we challenged mice lacking the GH receptor gene (Ghr(-/-), a model for GH resistance) by crossing them with Mdr2 knockout mice (Mdr2(-/-)), a mouse model of inflammatory cholestasis and liver fibrosis. Ghr(-/-);Mdr2(-/-) mice showed elevated serum markers associated with liver damage and cholestasis, extensive bile duct proliferation, and increased collagen deposition relative to Mdr2(-/-) mice, thus suggesting a more severe liver fibrosis phenotype. Additionally, Ghr(-/-);Mdr2(-/-) mice had a pronounced down-regulation of hepatoprotective genes Hnf6, Egfr, and Igf-1, and significantly increased levels of reactive oxygen species (ROS) and apoptosis in hepatocytes, compared to control mice. Moreover, single knockout mice (Ghr(-/-)) fed with a diet containing 1% cholic acid displayed an increase in hepatocyte ROS production, hepatocyte apoptosis, and bile infarcts compared to their wild-type littermates, indicating that loss of Ghr renders hepatocytes more susceptible to toxic bile acid accumulation. Surprisingly, and despite their severe fibrotic phenotype, Ghr(-/-);Mdr2(-/-) mice displayed a significant decrease in tumor incidence compared to Mdr2(-/-) mice, indicating that loss of Ghr signaling may slow the progression from fibrosis/cirrhosis to cancer in the liver. GH resistance dramatically exacerbates liver fibrosis in a mouse model of inflammatory cholestasis, therefore suggesting that GH resistance plays a causal role in the disease and provides a novel target for the development of liver fibrosis treatments.