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Programmed cell death receptor ligand-1 (PD-L1) expression, KRAS (KRASm) and EGFR (EGFRm) mutations may influence non-small cell lung cancer (NSCLC) prognosis. We aimed to evaluate PD-L1 expression, KRASm, and EGFRm and survival among stage III unresected NSCLC patients. Using Danish registries, we collected data on stage III unresected NSCLC patients diagnosed 2001–2012 and paraffin-embedded tumor tissue from pathology archives. We assessed PD-L1 expression in tumors and tumor-infiltrating immune cells (ICs) by immunohistochemistry ($$\ge$$ ≥ 1% threshold for PD-L1+). We genotyped KRAS and EGFR. Follow-up extended from 120 days post-diagnosis to death, emigration, or 31/12/2014. We computed median survival using Kaplan–Meier methods, and hazard ratios (HRs) using Cox regression associating the biomarkers with death, adjusting for confounders. Among 305 patients, 48% had adenocarcinoma; 38% squamous cell carcinoma. Forty-nine percent had PD-L1+ tumors—51% stage IIIA and 26% KRASm. Few (2%) patients had EGFRm. Median survival in months was 14.7 (95% CI = 11.8–17.9) and 13.4 (95% CI = 9.5–16.3) in PD-L1+ and PD-L1− tumors, respectively. KRASm was not associated with death (HR = 1.06, 95% CI = 0.74–1.51 versus wildtype). PD-L1+ tumors yielded a HR = 0.83 (95% CI = 0.63–1.10); PD-L1+ ICs a HR = 0.51 (95% CI = 0.39–0.68). Tumor expression of PD-L1 did not influence survival. PD-L1+ ICs may confer survival benefit in stage III unresected NSCLC patients.

Normal liver tissue is highly vulnerable towards irradiation, which remains a challenge in radiotherapy of hepatic tumours. Here, we examined the effects of radiation-induced liver injury on two specific liver functions and hepatocellular regeneration in a minipig model. Five Göttingen minipigs were exposed to whole-liver stereotactic body radiation therapy (SBRT) in one fraction (14 Gy) and examined 4–5 weeks after; five pigs were used as controls. All pigs underwent in vivo positron emission tomography (PET) studies of the liver using the conjugated bile acid tracer [N-methyl-11C]cholylsarcosine ([11C]CSar) and the galactose-analogue tracer [18F]fluoro-2-deoxy-d-galactose ([18F]FDGal). Liver tissue samples were evaluated histopathologically and by immunohistochemical assessment of hepatocellular mitosis, proliferation and apoptosis. Compared with controls, both the rate constant for secretion of [11C]CSar from hepatocytes into intrahepatic bile ducts as well as back into blood were doubled in irradiated pigs, which resulted in reduced residence time of [11C]CSar inside the hepatocytes. Also, the hepatic systemic clearance of [18F]FDGal in irradiated pigs was slightly increased, and hepatocellular regeneration was increased by a threefold. In conclusion, parenchymal injury and increased regeneration after whole-liver irradiation was associated with enhanced hepatobiliary secretion of bile acids. Whole-liver SBRT in minipigs ultimately represents a potential large animal model of radiation-induced liver injury and for testing of normal tissue protection methods.

Purpose The proliferation marker Ki-67 has been used as a prognostic marker to separate low- and high-risk breast cancer subtypes and guide treatment decisions for adjuvant chemotherapy. The association of Ki-67 with response to tamoxifen therapy is unclear. High-throughput automated scoring of Ki-67 might enable standardization of quantification and definition of clinical cut-off values. We hypothesized that digital image analysis (DIA) of Ki-67 can be used to evaluate proliferation in breast cancer tumors, and that Ki-67 may be associated with tamoxifen resistance in early-stage breast cancer. Patients and methods Here, we apply DIA technology from Visiopharm using a custom designed algorithm for quantifying the expression of Ki-67, in a case-control study nested in the Danish Breast Cancer Group clinical database, consisting of stages I, II, or III breast cancer patients of 35-69 years of age, diagnosed during 1985-2001, in the Jutland peninsula, Denmark. We assessed DIA-Ki-67 score on tissue microarrays (TMAs) from breast cancer patients in a case-control study including 541 ER-positive and 300 ER-negative recurrent cases and their non-recurrent controls, matched on ER-status, cancer stage, menopausal status, year of diagnosis, and county of residence. We used logistic regression to estimate odds ratios and associated 95% confidence intervals to determine the association of Ki-67 expression with recurrence risk, adjusting for matching factors, chemotherapy, type of surgery, receipt of radiation therapy, age category, and comorbidity. Results Ki-67 was not associated with increased risk of recurrence in tamoxifen-treated patients (ORadj =0.72, 95% CI 0.54, 0.96) or ER-negative patients (ORadj =0.85, 95% CI 0.54, 1.34). Conclusion Our findings suggest that Ki-67 digital image analysis in TMAs is not associated with increased risk of recurrence among tamoxifen-treated ER-positive breast cancer or ER-negative breast cancer patients. Overall, our findings do not support an increased risk of recurrence associated with Ki-67 expression.

Background Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that facilitates the adaptation of cancer cells to hypoxic conditions and may be prognostic of breast cancer recurrence. We evaluated the association of HIF-1α expression with breast cancer recurrence, and its association with timing of breast cancer recurrence. Methods In this population-based case-control study, we included women diagnosed with stage I–III breast cancer between 1985 and 2001, aged 35–69 years, registered in the Danish Breast Cancer Group. We identified 541 cases of breast cancer recurrence among women with estrogen receptor (ER)-positive disease who were treated with tamoxifen for at least 1 year (ER+ TAM+). We also enrolled 300 breast cancer recurrence cases among women with ER-negative disease, not treated with tamoxifen, who survived at least 1 year (ER−/TAM−). Controls were recurrence-free breast cancer patients at the time of case diagnosis, matched to recurrence cases on ER/TAM status, date of surgery, menopausal status, cancer stage, and county of residence. Expression of HIF-1α was measured by immunohistochemistry on tissue microarrays. We fitted logistic regression models to compute odds ratios (ORs) and 95% confidence intervals (CIs) associating HIF-1α expression with recurrence, and with timing of recurrence. Results HIF-1α expression was observed in 23% of cases and 20% of controls in the ER+/TAM+ stratum, and in 47% of cases and 48% of controls in the ER−/TAM− stratum. We observed a near-null association between HIF-1α expression in both ER/TAM groups (ER+/TAM+ OR = 1.21, 95%CI 0.88, 1.67 and ER−/TAM− OR = 0.97, 95%CI 0.68, 1.39). HIF-1α expression was not associated with time to recurrence among women in the ER+/TAM+ stratum, but was associated with early recurrence among women in the ER−/TAM− stratum. Conclusion In this study, HIF-1α expression was not associated with breast cancer recurrence overall but may be associated with early recurrence among women diagnosed with ER− breast cancer.

BACKGROUND: PD-L1 expression on tumor cells (TCs) or immune cells (ICs) may be used as a prognostic marker for survival in patients with NSCLC. We characterized PD-L1 expression on TCs or ICs in a patient cohort with NSCLC to determine associations between PD-L1 expression and overall survival (OS), according to EGFR and KRAS mutation status.METHODS: Danish patients aged >18 years diagnosed with NSCLC before 2014 on first- (N = 491), second- (N = 368), or third-line (N = 498) therapy were included. Data were extracted from population-based medical registries. Tumor samples from pathology archives were tested for biomarkers. High PD-L1 expression was defined as expression on ≥25 % of TCs or ICs based on first diagnostic biopsy or surgical resection. KRAS and EGFR mutation status were tested using PCR-based assays. Cox regression analysis was used to compute adjusted HRs and associated 95 % CIs.RESULTS: PD-L1 TC and IC ≥ 25 % were observed in 24.3 %-31.0 % and 11.7-14.7 % of patients, respectively. EGFR and KRAS mutations were detected in 4.7 %-8.8 % and 26.5 %-30.7 % of patients, respectively. PD-L1 TC ≥ 25 % was not associated with survival advantage in first- (HR = 0.96, 95 % CI: 0.75-1.22), second- (1.08, 0.81-1.42), or third-line (0.94, 0.74-1.20) therapy. PD-L1 IC ≥ 25 % was associated with survival advantage in second-line (HR = 0.56, 95 % CI: 0.36-0.86) and third-line (0.69, 0.49-0.97) but not first-line (1.00, 0.70-1.41) therapy.CONCLUSION: No association was observed between PD-L1 TC ≥ 25 % and OS in any therapy line. PD-L1 IC ≥ 25 % may confer survival benefit among some patients who reach second-line therapy.

Our study provides novel findings of experimental hypokalemia reducing urea cycle functionality and thereby severely increasing plasma ammonia. This is pathophysiologically interesting because plasma ammonia increases during hypokalemia by a hitherto unknown mechanism, which may be particular important in relation to the unexplained link between hypokalemia and hepatic encephalopathy. Potassium deficiency decreases gene expression, protein synthesis, and growth. The urea cycle maintains body nitrogen homeostasis including removal of toxic ammonia. Hyperammonemia is an obligatory trait of liver failure, increasing the risk for hepatic encephalopathy, and hypokalemia is reported to increase ammonia. We aimed to clarify the effects of experimental hypokalemia on the in vivo capacity of the urea cycle, on the genes of the enzymes involved, and on ammonia concentrations. Female Wistar rats were fed a potassium-free diet for 13 days. Half of the rats were then potassium repleted. Both groups were compared with pair- and free-fed controls. The following were measured: in vivo capacity of urea-nitrogen synthesis (CUNS); gene expression (mRNA) of urea cycle enzymes; plasma potassium, sodium, and ammonia; intracellular potassium, sodium, and magnesium in liver, kidney, and muscle tissues; and liver sodium/potassium pumps. Liver histology was assessed. The diet induced hypokalemia of 1.9 ± 0.4 mmol/L. Compared with pair-fed controls, the in vivo CUNS was reduced by 34% ( P < 0.01), gene expression of argininosuccinate synthetase 1 ( ASS1) was decreased by 33% ( P < 0.05), and plasma ammonia concentrations were eightfold elevated ( P < 0.001). Kidney and muscle tissue potassium contents were markedly decreased but unchanged in liver tissue. Protein expressions of liver sodium/potassium pumps were unchanged. Repletion of potassium reverted all the changes. Hypokalemia decreased the capacity for urea synthesis via gene effects. The intervention led to marked hyperammonemia, quantitatively explainable by the compromised urea cycle. Our findings motivate clinical studies of patients with liver disease.

Post-transplant lymphoproliferative disorders (PTLDs) are lymphoid proliferations that can arise as a complication to organ transplantation [1,2]. When compared with sporadically occurring lymphoma...

Marie Beck Enemark,1,2,* Ida Monrad,1,* Charlotte Madsen,1 Kristina Lystlund Lauridsen,3 Bent Honoré,4 Trine Lindhardt Plesner,5 Stephen Jacques Hamilton-Dutoit,3 Francesco d’Amore,1 Maja Ludvigsen1,2 1Department of Hematology, Aarhus University Hospital, Aarhus, Denmark; 2Department of Clinical Medicine, Aarhus University, Aarhus, Denmark; 3Department of Pathology, Aarhus University Hospital, Aarhus, Denmark; 4Department of Biomedicine, Aarhus University, Aarhus, Denmark; 5Department of Pathology, Copenhagen University Hospital, Copenhagen, Denmark*These authors contributed equally to this workCorrespondence: Maja LudvigsenDepartment of Hematology, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, C115, Aarhus N 8200, DenmarkTel +45 22859523Email majlud@rm.dkPurpose: Follicular lymphoma (FL) is an indolent, yet generally incurable neoplasia with a median survival exceeding 10 years. However, a subset of FL patients experiences histological transformation (HT) to a more aggressive lymphoma, in the majority of cases to diffuse large B-cell lymphoma (DLBCL). This affects both the clinical course and the prognostic outcome, resulting in a markedly reduced survival after transformation. Thus, early risk stratification and prediction of patients at risk of HT would be highly valuable in the clinical setting. Here, we investigated the potential of the immune inhibitory programmed death 1 (PD-1) receptor as a biomarker predictive of HT.Patients and Methods: Immunohistochemical staining and quantification by digital image analysis of PD-1 was performed on diagnostic tumor-tissue samples from FL patients with and without subsequent transformation (n=34 and n=46, respectively), and on paired samples from the transformed lymphoma (n=34).Results: At the time of initial FL diagnosis, samples from patients with subsequent HT had significantly higher tumor-tissue expression of PD-1 compared with diagnostic FL samples from patients without subsequent HT (p=0.010). At the time of transformation, PD-1 expression was significantly reduced (p< 0.001). No difference was observed in intra-follicular PD-1 expression at FL diagnosis between samples from patients with or without HT; however, high intra-follicular levels of PD-1 were associated with significantly shorter transformation-free survival times (p< 0.043).Conclusion: Our data suggest that pre-treatment tumor-tissue PD-1 expression already predicts the risk of subsequent transformation to DLBCL, as early as the time of FL diagnosis.Keywords: programmed death 1; PD-1, follicular lymphoma; FL, histological transformation; HT

Background: Statins treat hyperlipidemia and prevent cardiovascular morbidity and mortality. Evidence suggests that they also have anti-neoplastic activity. Several studies show a reduced rate of breast cancer recurrence among lipophilic statin users (e.g., simvastatin), motivating calls for clinical trials of statins in breast cancer patients. We measured the impact of genetic variation in statin-metabolizing enzymes and drug transporters on the recurrence rate in simvastatin-treated breast cancer patients. Methods: We conducted a nested case-control study among Danish women diagnosed with non-metastatic, invasive breast cancer between 2004–2010 who had filled ≥1 prescription for simvastatin after diagnosis. Cases were all breast cancer recurrences from the source population; one control was matched to each case on cancer stage, estrogen receptor and hormone therapy status, calendar period of diagnosis, and duration of simvastatin exposure. We genotyped variants in simvastatin-metabolizing enzymes (CYP3A4/rs35599367 and CYP3A5/rs776746) and drug transporters (ABCB1/rs2032582 and SLCO1B1/rs4149056), and estimated their association with recurrence with logistic regression models. Results: We observed protective (though imprecisely-measured) associations between variants in genes encoding drug transporters (ABCB1 and SLCO1B1) and simvastatin-metabolizing enzymes (CYP3A4 and CYP3A5) and breast cancer recurrence in simvastatin-treated women. For example, carrying two variant alleles in ABCB1 was associated with a 31% lower rate of recurrence (multivariable OR = 0.69, 95% CI: 0.31, 1.5). Conclusion: Our study provides weak evidence to support the use of genetic variation in ABCB1, SLCO1B1, CYP3A4, and CYP3A5 as biomarkers of breast tumor response to simvastatin. Validation of these findings within adjuvant clinical trials is encouraged.

INTRODUCTION: In alcoholic hepatitis (AH), high interleukin (IL)-22 production is associated with disease improvement, purportedly through enhanced infection resistance and liver regeneration. IL-22 binding protein (BP) binds and antagonizes IL-22 bioactivity, but data on IL-22BP in liver disease suggest a complex interplay. Despite the scarcity of human data, IL-22 is in clinical trial as treatment of AH. We, therefore, in patients with AH, described the IL-22 system focusing on IL-22BP and associations with disease course, and mechanistically pursued the human associations in vitro.METHODS: We prospectively studied 41 consecutive patients with AH at diagnosis, days 7 and 90, and followed them for up to 1 year. We measured IL-22 pathway proteins in liver biopsies and blood and investigated IL-22BP effects on IL-22 in hepatocyte cultures.RESULTS: IL-22BP was produced in the gut and was identifiable in the patients with AH' livers. Plasma IL-22BP was only 50% of controls and the IL-22/IL-22BP ratio thus elevated. Consistently, IL-22-inducible genes were upregulated in AH livers at diagnosis. Low plasma IL-22BP was closely associated with high 1-year mortality. In vitro, IL-22 stimulation reduced IL-22 receptor (R) expression, but coincubation with IL-22BP sustained IL-22R expression. In the AH livers, IL-22R mRNA expression was similar to healthy livers, although IL-22R liver protein was higher at diagnosis.DISCUSSION: Plasma IL-22BP was associated with an adverse disease course, possibly because its low level reduces IL-22R expression so that IL-22 bioactivity was reduced. This suggests the IL-BP interplay to be central in AH pathogenesis, and in future treatment trials (see Visual abstract, Supplementary Digital Content 5, http://links.lww.com/CTG/A338).

Background: Tamoxifen and its metabolites compete with estrogen to occupy the estrogen receptor. The conventional dose of adjuvant tamoxifen overwhelms estrogen in this competition, reducing breast cancer recurrence risk by nearly half. Phase I metabolism generates active tamoxifen metabolites, and phase II metabolism deactivates them. No earlier pharmacogenetic study has comprehensively evaluated the metabolism and transport pathways, and no earlier study has included a large population of premenopausal women. Methods: We completed a cohort study of 5,959 Danish nonmetastatic premenopausal breast cancer patients, in whom 938 recurrences occurred, and a case–control study of 541 recurrent cases in a cohort of Danish predominantly postmenopausal breast cancer patients, all followed for 10 years. We collected formalin-fixed paraffin-embedded tumor blocks and genotyped 32 variants in 15 genes involved in tamoxifen metabolism or transport. We estimated conventional associations for each variant and used prior information about the tamoxifen metabolic path to evaluate the importance of metabolic and transporter pathways. Results: No individual variant was notably associated with risk of recurrence in either study population. Both studies showed weak evidence of the importance of phase I metabolism in the clinical response to adjuvant tamoxifen therapy. Conclusions: Consistent with prior knowledge, our results support the role of phase I metabolic capacity in clinical response to tamoxifen. Nonetheless, no individual variant substantially explained the modest phase I effect on tamoxifen response. Impact: These results are consistent with guidelines recommending against genotype-guided prescribing of tamoxifen, and for the first time provide evidence supporting these guidelines in premenopausal women.

BACKGROUND: Approximately 20–40% of patients diagnosed with breast cancer will experience a recurrence up to 20 years after their original diagnosis. 17?-hydroxysteroid dehydrogenase 1 and 2 (HSD17B1 and HSD17B2, respectively) regulate intratumoral concentrations of oestradiol, which promotes growth and proliferation of hormone dependent tumours. Breast carcinomas with increased HSD17B1, without a corresponding increase in HSD17B2, expression may become resistant to tamoxifen therapy by producing locally higher concentrations of oestradiol, which compete with tamoxifen and its metabolites for binding to the oestrogen receptor (ER). MATERIALS AND METHODS: In this population-based case-control study, we included women diagnosed with stage I–III breast cancer between 1985 and 2001, aged 35–69 years, registered in the Danish Breast Cancer Group. We identified 541 cases of breast cancer recurrence among women with ER positive disease who were treated with tamoxifen for at least 1 year (ER+TAM+). We also enrolled 300 breast cancer recurrence cases among women with ER negative disease, not treated with tamoxifen, who survived at least 1 year (ER?/TAM?). Controls were recurrence-free breast cancer patients at the time of case diagnosis, matched to recurrence cases on ER/TAM status, date of surgery, menopausal status, stage, and county or residence. Expression of HSD17B1 and HSD17B2 were measured by immunohistochemistry on tissue microarrays. We fit logistic regression models to compute odds ratios (ORs) and 95% confidence intervals (CIs) associating the HSD17B1:HSD17B2 ratio (>1 vs. ?1)—and each enzyme’s independent expression—with recurrence. RESULTS: We found no association between the HSD17B1:HSD17B2 ratio and breast cancer recurrence in either ER/TAM stratum (ER+/TAM+: OR=1.03, 95% CI: 0.78, 1.40; ER?/TAM?: OR=1.02, 95% CI: 0.77, 1.19). Associations for expression of each individual enzyme were also near null. CONCLUSION: The ratio of HSD17B1 expression to HSD17B2 expression was not associated with breast cancer recurrence in this study.

Galectin-1 (Gal-1) has been associated with adverse prognosis in several cancers including lymphoma entities with CD30 expression. However, Gal-1 expression has not been systematically assessed in peripheral T-cell lymphomas (PTCL). Specimens from 169 nodal PTCL were assessed for intratumoural Gal-1 expression by immunohistochemistry. Overall survival (OS) in groups exhibiting high and low Gal-1 expression was compared in the cohort and in a subset analysis of CD30-positive PTCL only. Gal-1 expression was also correlated with biomarkers of the tumour microenvironment. No significant difference in OS based on Gal-1 expression was observed in the entire PTCL cohort. However, in the CD30-positive cohort, patients with high Gal-1 levels had significantly poorer outcome (5 years OS 10%, 95% confidence interval CI, 1-36) than their low Gal-1 counterparts (5 years OS 48%, 95% CI, 30-64, P =.021). In univariate analyses age 60 or younger, non-elevated lactate dehydrogenase (LDH), and performance score less than 2 correlated with superior survival but high Gal-1 expression significantly predicted adverse outcome at both univariate (HR 2.5, 95% CI, 1.1-5.7, P =.026) and multivariate levels (HR 3.2, 95% CI, 1.2-8.5, P =.017). Tumours with high Gal-1 had few cytotoxic T cells in the tumour microenvironment. High intratumoural Gal-1 expression before therapeutic intervention correlates with adverse outcome in nodal CD30+, ALK− PTCL patients.

Myeloid and lymphoid malignancies are postulated to have distinct pathogenic mechanisms. The recent observation that patients with a myeloproliferative neoplasm have an increased risk of developing lymphoproliferative malignancies has challenged this assumption. We collected a nationwide cohort of patients with both malignancies. Patients diagnosed between 1990 and 2015 were identified through the national Danish Pathology Registry. We identified 599 patients with a myeloproliferative neoplasm and a concomitant or subsequent diagnosis of lymphoma. Histopathological review of the diagnostic samples from each patient led to a final cohort of 97 individuals with confirmed dual diagnoses of myeloproliferative neoplasm and lymphoma. The age range at diagnosis of these individuals was 19-94 years (median: 71 years). To avoid the inclusion of cases of therapy-induced myeloproliferative neoplasm occurring in patients previously treated for lymphoma, only patients with myeloproliferative neoplasm diagnosed unequivocally before the development of lymphoma were included. The average time interval between the diagnoses of the two malignancies was 1.5 years. In the majority of patients (90%) both diagnoses were established within 5 years of each other. Among the lymphoma entities, the frequency of peripheral T-cell lymphomas was markedly increased. Interestingly, all but one of the T-cell lymphomas were of angioimmunoblastic type. These findings suggest that a myeloproliferative neoplasm and lymphoproliferative malignancy developing in the same patient may have common pathogenic events, possibly already at the progenitor level. We believe that the molecular characterization of the newly developed biorepository will help to highlight the mechanisms driving the genesis and clonal evolution of these hematopoietic malignancies.

Background: Non-alcoholic fatty liver disease (NAFLD) is a major health concern affecting 25% of the world's population. It is generally held that a fatty liver does not influence liver function, but quantitative measurements of metabolic liver functions have not been systematically performed. We aimed to study selected hepatocellular metabolic functions in patients with different stages of NAFLD. Methods: Twenty-five non-diabetic, biopsy-proven NAFLD patients [12 with simple steatosis; 13 with non-alcoholic steatohepatitis (NASH)] and ten healthy controls were included in a cross-sectional study. Hepatocyte cytosolic function was assessed by the galactose elimination capacity (GEC), mitochondrial-cytosolic metabolic capacity by the functional hepatic nitrogen clearance (FHNC), microsomal function by the aminopyrine breath test, and excretory liver function by indocyanine green (ICG) elimination. Results: GEC was 20% higher in NAFLD than in controls [3.15 mmol/min (2.9–3.41) vs. 2.62 (2.32–2.93); P = 0.02]. FHNC was 30% lower in NAFLD [23.3 L/h (18.7–28.9) vs. 33.1 (28.9–37.9); P = 0.04], more so in simple steatosis [19.1 L/h (13.9–26.2); P = 0.003] and non-significantly in NASH [27.9 L/h (20.6–37.8); P = 0.19]. Aminopyrine metabolism was 25% lower in simple steatosis [8.9% (7.0–10.7)] and 50% lower in NASH [6.0% (4.5–7.5)] than in controls [11.9% (9.3–12.8)] (P < 0.001). ICG elimination was intact. Conclusions: The hepatocellular metabolic functions were altered in a manner that was dissociated both by different effects on different liver functions and by different effects of different stages of NAFLD. Thus, NAFLD has widespread consequences for metabolic liver function, even in simple steatosis.

A 37-year-old male, who at the age of 8 years had been treated for right-sided Wilms’ tumor with nephrectomy, radiotherapy, and chemotherapy, presented with noncirrhotic portal hypertension (NCPH), grade 2 esophageal varices, and ascites. A CT scan demonstrated hypoplasia of liver segments 2 and 3. A liver biopsy showed portal tract fibrosis without cirrhosis, with histological features of NCPH. Liver vein catheterization showed a normal portal pressure gradient of 5 mm Hg while spleen to hepatic vein pressure was 29 mm Hg. NCPH after therapy for Wilms’ tumor is described in children within the first few years after treatment. This is the first case report in which the patient first presented symptoms as an adult, many years after cancer treatment.

Background and aim Liver macrophages are activated in chronic hepatitis B virus (CHB) infection and play a pivotal role in hepatic inflammation and fibrosis. However, their role during anti-viral treatment is unclear. The soluble (s) macrophage activation markers, sCD163 and mannose receptor (sMR), are released during liver damage and their serum levels reflect liver disease severity and portal hypertension. We aimed to investigate associations between sCD163 and sMR and histopathological activity and fibrosis, and changes in sCD163, sMR and hepatic CD163-expression following anti-viral treatment in CHB patients. Methods We assessed Ishak histological necroinflammatory activity and fibrosis scores in liver biopsies from 254 CHB patients, and serially in 71 patients before and after nucleoside-analogue treatment. Liver CD163-expression was semi-quantitatively determined by immunohistochemistry and serum sCD163 and sMR measured by ELISA. Results Before treatment, the mean levels of sCD163 and sMR were 3.57 (SD 1.72) mg L-1 and 0.35 (0.12) mg L-1. sCD163 and sMR increased with histological inflammatory activity (sCD163: r=0.46, p

Purpose Our aim was to capture the biology of classical Hodgkin lymphoma (cHL) at the time of relapse and discover novel and robust biomarkers that predict outcomes after autologous stem-cell transplantation (ASCT). Materials and Methods We performed digital gene expression profiling on a cohort of 245 formalin-fixed, paraffin-embedded tumor specimens from 174 patients with cHL, including 71 with biopsies taken at both primary diagnosis and relapse, to investigate temporal gene expression differences and associations with post-ASCT outcomes. Relapse biopsies from a training cohort of 65 patients were used to build a gene expression–based prognostic model of post-ASCT outcomes (RHL30), and two independent cohorts were used for validation. Results Gene expression profiling revealed that 24% of patients exhibited poorly correlated expression patterns between their biopsies taken at initial diagnosis and relapse, indicating biologic divergence. Comparative analysis of the prognostic power of gene expression measurements in primary versus relapse specimens demonstrated that the biology captured at the time of relapse contained superior properties for post-ASCT outcome prediction. We developed RHL30, using relapse specimens, which identified a subset of high-risk patients with inferior post-ASCT outcomes in two independent external validation cohorts. The prognostic power of RHL30 was independent of reported clinical prognostic markers (both at initial diagnosis and at relapse) and microenvironmental components as assessed by immunohistochemistry. Conclusion We have developed and validated a novel clinically applicable prognostic assay that at the time of first relapse identifies patients with unfavorable post-ASCT outcomes. Moving forward, it will be critical to evaluate the clinical use of RHL30 in the context of positron emission tomography–guided response assessment and the evolving cHL treatment landscape.

Immune checkpoint inhibition targeting the programmed death receptor (PD)-1 pathway is a novel treatment approach in relapsed and refractory classical Hodgkin lymphoma (cHL). Identifying patients with a high risk of treatment failure could support the use of PD-1 inhibitors as front-line treatment. Our aim was to investigate the prognostic impact of PD-1, programmed death-ligand 1 (PD-L1), and PD-L2 in the tumor microenvironment in diagnostic biopsies of patients with cHL. Patients from Denmark and Sweden, diagnosed between 1990 and 2007 and ages 15 to 86 years, were included. Tissue microarray samples were available from 387 patients. Immunohistochemistry was used to detect PD-1, PD-L1, and PD-L2, and the proportions of positive cells were calculated. Event-free survival (EFS; time to treatment failure) and overall survival (OS) were analyzed using Cox proportional hazards regression. High proportions of both PD-1

To the editor: Peripheral T-cell lymphomas (PTCLs) represent a group of rare hematological cancers of mature T-cell or natural killer cell origin accounting for 10% to 15% of all lymphomas.[1][1] Although many patients have poor outcomes, some achieve long-term survival.[2][2],[3][3] Thus

Key Clinical Message We report an otherwise healthy 32-year-old man with portal hypertension, variceal bleeding, and congenital hepatic fibrosis with ductal plate malformation. Genetic screening identified two TMEM67 mutations. Biallelic TMEM67 mutations are known to cause Joubert/Meckel syndrome or nephronopthisis with hepatic fibrosis, but have never been found in isolated hepatic fibrosis.

Tamoxifen therapy for estrogen receptor-positive breast cancer reduces the risk of recurrence by approximately one-half. Cytochrome P-450 2D6, encoded by the polymorphic cytochrome P-450 2D6 gene (CYP2D6), oxidizes tamoxifen to its most active metabolites. Steady-state concentrations of endoxifen (4-hydroxy-N-desmethyltamoxifen), the most potent antiestrogenic metabolite, are reduced in women whose CYP2D6 genotypes confer poor enzyme function. Thirty-one studies of the association of CYP2D6 genotype with breast cancer survival have yielded heterogeneous results. Some influential studies genotyped DNA from tumor-infiltrated tissues, and their results may have been susceptible to germline genotype misclassification from loss of heterozygosity at the CYP2D6 locus. We systematically reviewed 6 studies of concordance between genotypes obtained from paired nonneoplastic and breast tumor-infiltrated tissues, all of which showed excellent CYP2D6 genotype agreement. We applied these concordance data to a quantitative bias analysis of the subset of the 31 studies that were based on genotypes from tumor-infiltrated tissue to examine whether genotyping errors substantially biased estimates of association. The bias analysis showed negligible bias by discordant genotypes. Summary estimates of association, with or without bias adjustment, indicated no clinically important association between CYP2D6 genotype and breast cancer survival in tamoxifen-treated women.

Aims: Metformin is first-line treatment of type 2 diabetes mellitus and reduces cardiovascular events in patients with insulin resistance and type 2 diabetes. Target tissue for metformin action is thought to be the liver, where metformin distribution depends on facilitated transport by polyspecific transmembrane organic cation transporters (OCTs). Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the western world with strong associations to insulin resistance and the metabolic syndrome, but whether NAFLD affects metformin biodistribution to the liver is not known. In this study, the primary aim was to investigate in vivo hepatic uptake of metformin dynamically in humans with variable degrees of liver affection. As a secondary aim, we wished to correlate hepatic metformin distribution with OCT gene transcription determined in diagnostic liver biopsies. Methods: Eighteen patients with biopsy-proven NAFLD were investigated using 11C-metformin PET/CT technique. Gene transcripts of OCTs were determined by real-time polymerase chain reaction (PCR). Results: We observed similar hepatic volume of distribution of metformin between patients with simple steatosis and non-alcoholic steatohepatitis (NASH) (Vd 2.38 ± 0.56 vs. 2.10 ± 0.39, P = 0.3). There was no association between hepatic exposure to metformin and the degree of inflammation or fibrosis, and no clear correlation between metformin distribution and OCT gene transcription. Conclusion: Metformin is distributed to the liver in patients with NAFLD and the distribution is not impaired by inflammation or fibrosis. The findings imply that metformin action in liver in patients with NAFLD may be preserved.

Background: Adjuvant tamoxifen therapy approximately halves the risk of recurrence in estrogen receptor-positive (ER+) breast cancer patients, but many women respond insufficiently to therapy. Expression of multi-drug resistance protein 2 (MRP2) in breast cancer may potentiate tamoxifen resistance. Thus, we investigated the expression of MRP2 in breast cancer as a predictor of tamoxifen therapy effectiveness. Material and methods: We conducted a case-control study nested in the Danish Breast Cancer Group clinical database. The study included women aged 35–69 years diagnosed with stage l–lll breast cancer during 1985–2001, in Jutland, Denmark. We identified 541 recurrent breast cancers (cases) among women with estrogen receptor positive (ER+) disease treated with tamoxifen for at least 1 year (ER+/TAM+) and 300 cases among women with estrogen receptor-negative (ER−) disease, never treated with tamoxifen (ER−/TAM−). We matched one recurrence-free control to each recurrent case. We retrieved paraffin-embedded primary tumor tissue for all patients, and all available recurrent tumor tissue from pathology archives. MRP2 expression was evaluated using immunohistochemistry. We computed odds ratios (ORs) and 95% confidence intervals (95% CIs) associating MRP2 expression (positive vs. none) with breast cancer recurrence in conditional logistic regression models. We compared MRP2 expression in paired primary- and recurrent tumors. Results: MRP2 expression was more prevalent in the ER+/TAM + group, than in the ER−/TAM − group. No predictive utility of MRP2 for breast cancer recurrence was found in the ER+/TAM + group (ORadj = 0.96, 95% CI 0.70, 1.33). Further, no prognostic utility was found in the ER−/TAM − group (ORadj = 0.81, 95% CI 0.53, 1.23). MRP2 expression was not increased in recurrent versus primary tumors. Conclusions: MRP2 expression is neither a predictive marker of tamoxifen effectiveness nor a prognostic marker in breast cancer.

18F-fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (FDG-PET/CT) is used for staging classical Hodgkin lymphoma (cHL) with high sensitivity for skeletal involvement. However, it is unclear whether a single bone lesion carries the same adverse prognosis as multifocal lesions and if this is affected by type of chemotherapy [ABVD (adriamycin, bleomycin, vincristine, dacarbazine) versus BEACOPP (bleomycin, etoposide, adriamycin, cyclophosphamide, vincristine, procarbazine, prednisone)]. We reviewed the clinico-pathological and outcome data from 209 patients with newly diagnosed cHL staged by FDG-PET/CT. Patterns of skeletal/bone marrow uptake (BMU) were divided into ‘low’ and ‘high’ diffuse BMU (i.e. without focal lesions), and unifocal or multifocal lesions. Additional separate survival analysis was performed, taking type of chemotherapy into account. Forty patients (19·2%) had skeletal lesions (20 unifocal, 20 multifocal). The 3-year progression-free-survival (PFS) was 80% for patients with ‘low BMU’, 87% for ‘high BMU’, 69% for ‘unifocal’ and 51% for ‘multifocal’ lesions; median follow-up was 38 months. The presence of bone lesions, both uni- and multifocal, was associated with significantly inferior PFS (log rank P = 0·0001), independent of chemotherapy type. Thus, increased diffuse BMU should not be considered as a risk factor in cHL, whereas unifocal or multifocal bone lesions should be regarded as important predictors of adverse outcome, irrespective of the chemotherapy regimen used.

At AHIV-1 infection, the binding of the viral envelopeproteins to CD4þ is essential for viral transmission, andthis process is facilitated by interaction with the highlyconserved host lectin, galectin-1 (Gal-1) [1?3]. Withinthe tumor microenvironment, Gal-1 is expressed by bothtumor and stromal cells where it promotes tumorimmune escape and favors hypoxia-driven angiogenesis[4?6]. In sporadically occurring Hodgkin lymphoma,high Gal-1 expression at diagnosis is associated withpoorer treatment response [7], and high soluble Gal-1(sGal-1) correlates with adverse disease characteristics [8].Previous studies have shown that targeted inhibition ofGal-1 prevents tumor-induced immunosuppression[9,10] as well as inhibits tumor growth and metastasisin various tumor models [6,11?13].In conclusion, the results of our study indicate that Gal-1 is significantly associated with risk of lymphoma in HIV-infected individuals and may represent an attractive futuretarget for the management of HIV-associated lymphoma. Fil: Vase, MajaØlholm. University Aarhus; Dinamarca Fil: Ludvigsen, Maja. University Aarhus; Dinamarca Fil: Bendix, Knud. University Aarhus; Dinamarca Fil: Dutoit, Stephen H.. University Aarhus; Dinamarca Fil: Hjortebjerg, Rikke. University Aarhus; Dinamarca Fil: Petruskevicius, Irma. University Aarhus; Dinamarca Fil: Møller, Michael B.. Odense Universitetshospital; Dinamarca Fil: Pedersen, Gitte. Aalborg Universitet; Dinamarca Fil: Denton, Paul W.. University Aarhus; Dinamarca. Arhus Universitetshospital; Dinamarca Fil: Honoré, Bent. University Aarhus; Dinamarca Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Larsen, Carsten S.. Arhus Universitetshospital; Dinamarca Fil: D'Amore, Francesco. Arhus Universitetshospital; Dinamarca

BACKGROUND: Autoimmune hepatitis (AIH) is characterised by liver inflammation with reversibility upon anti-inflammatory treatment. Soluble (s)CD163, a specific macrophage activation marker, is associated with inflammation in other liver diseases, but never investigated in AIH.AIM: To investigate sCD163 in patients with acute AIH and in complete and incomplete responders to standard anti-inflammatory pharmacotherapy, and during follow-up in treatment naive patients.METHODS: In a cross-sectional design, we studied 121 AIH patients (female/male 89/32, median age 49 years); of these, we prospectively studied 10 treatment naïve AIH patients during prednisolone treatment and tapering. Twenty patients had variant syndromes of AIH and primary biliary cholangitis or primary sclerosing cholangitis. sCD163 was compared with markers of disease activity, severity and treatment response.RESULTS: In the patients with acute AIH (n = 21), sCD163 was sixfold increased compared with the normalised levels in patients (n = 32) with complete response to standard treatment [9.5 (3.3-28.8) vs. 1.6 (0.8-3.2) mg/L, P < 0.01)], while the patients (n = 27) with incomplete response had higher sCD163 [2.2 (1.3-7.9), P < 0.05] than the complete responders. sCD163 was positively associated with ALAT, IgG and bilirubin (rho: 0.45-0.59, P < 0.001, all), and negatively to external coagulation factors (rho:-0.34, P < 0.001). In the treatment naïve patients, sCD163 fell during high-dose prednisolone treatment and tapering. Immunohistochemical staining confirmed increased CD163 expression in liver biopsies from patients with acute AIH.CONCLUSIONS: sCD163 was markedly elevated in AIH in the acute phase, normalised by successful treatment in complete responders, but remained higher in the incompletely responding cases. Our results demonstrate macrophage activation in AIH paralleling disease activity, severity and treatment response, suggesting a role for macrophage activation in AIH.

BACKGROUND: The upper limit for the size of hepatectomy is approximately 90% in rats. The aim of the study was to assess quantitatively using stereological methods the impact on liver function, regeneration rate (RR), and hepatocyte proliferation of varying hepatectomy size in a rat model.MATERIALS AND METHODS: A total of 104 male Wistar rats were subjected to 30%, 70%, or 90% partial hepatectomy, sham operation, or no operation. Euthanization and harvesting of liver tissue and blood took place at postoperative days 1, 3, and 5 (n = 8 per group). Liver-specific biochemistry and RR were evaluated. Hepatocyte proliferation was estimated by immunohistochemical staining for Ki-67 antigen using unbiased stereological principles.RESULTS: Liver RR in the 90% group increased by a 6.6 fold during the 5 postoperative days compared with only a minor increase in both the 70% and 30% partial hepatectomy groups. The highest number of Ki-67-positive hepatocytes was observed in the 70% group at postoperative day 1 and for the 90% group at postoperative day 3. Prothrombin-proconvertin ratio was significantly lower in the 90% group 1 d after surgery compared with all other groups, however, nearly normalized at postoperative day 5.CONCLUSIONS: We show that liver RR and the number of proliferating hepatocytes increase, whereas the initial hepatic synthetic capacity decreases with increasing hepatectomy size.

Objective It has been hypothesized that Lewy pathology initiates in the enteric nervous system years prior to debut of clinical motor symptoms in Parkinson disease patients. This study investigates whether Lewy pathology is present in various gastrointestinal tract tissues from Parkinson disease patients in the prodromal phase. Methods We used the Danish National Pathology Registry to identify archived paraffin-embedded tissue blocks from 57 Parkinson disease patients (98 blocks) and 90 control subjects (98 blocks). We employed 2 different immunohistochemistry techniques visualizing aggregated α-synuclein and phosphorylated α-synuclein. Results Thirty-nine Parkinson disease patients contributed tissues obtained in the prodromal disease phase, whereas 18 Parkinson disease patients contributed tissues obtained solely after Parkinson diagnosis. Prodromal tissues were obtained on average 7.0 years prior to diagnosis (range = 20 years to 4 months), and postdiagnosis tissue on average 2.8 years after diagnosis (range = 2 days to 18 years). Phosphorylated α-synuclein positivity was seen in 22 of 39 (56%) prodromal Parkinson disease subjects and 30 of 67 (45%) prodromal tissue blocks. These fractions were significantly higher compared to control subjects (p = 0.0001 and p = 0.0032, respectively). In contrast, no significant difference was seen in the positivity rate between prodromal Parkinson disease patients and controls when using the aggregated α-synuclein immunohistochemistry technique. Interpretation We detected Lewy pathology in the gastrointestinal tract of patients up to 20 years prior to their Parkinson disease diagnosis. These findings are in accordance with a hypothesized prodromal disease phase spanning 10 to 20 years. Ann Neurol 2016;79:940–949

Background: Tamoxifen therapy for estrogen receptor positive (ER+) breast cancer reduces recurrence risk by about half. Steady-state concentrations of endoxifen, a potent anti-estrogenic tamoxifen metabolite, are reduced in women whose CYP2D6 genotypes confer poor enzyme function. Many studies have measured associations between genetically impaired CYP2D6 function and tamoxifen resistance. It has been suggested that the subset of studies using DNA extracted from tumor-infiltrated tissue may have been susceptible to genotyping error induced by loss of heterozygosity (LOH); the putative non-differential genotype misclassification may have biased these studies' estimates toward the null. We reviewed the clinical epidemiology studies conducted to date to assess the importance of loss-of-heterozygosity (LOH) at the CYP2D6 locus and its implications for assessing tamoxifen effectiveness. Methods: We searched for the terms "tamoxifen" and "CYP2D6" in PubMed, including all papers and abstracts through 31 May 2015 on the association of CYP2D6 gene variants and the risk of breast cancer recurrence or mortality. We used a quantitative bias analysis (QBA) to evaluate the importance of genotype misclassification in studies that extracted DNA from tumor-infiltrated tissue. We conducted a random effects meta-analysis to evaluate all studies simultaneously, and within groups according to whether DNA was derived from tumor-infiltrated tissue or non-neoplastic tissue. Results: Thirty-one studies investigated CYP2D6 genotype and breast cancer recurrence, yielding relative effect estimates ranging from 0.08 to 14. DNA was extracted from blood or non-neoplastic tissue in 21 of these 31 studies (68%), and from tumor-infiltrated tissue in the remaining 10 (32%). Our analysis of the association between variant/variant genotype compared with wildtype/wildtype genotype included 21 of the 31 studies. Sixteen (76%) of these 21 studies extracted DNA from blood or non-neoplastic tissue and five (24%) extracted DNA from tumor-infiltrated tissue. Genotype misclassification parameters for the QBA were estimated from six concordance studies. There was little difference between the effect estimates (EE) and 95% confidence/simulation intervals (95% CI/SI) before and after QBA (EE=1.71, 95%CI=1.24, 2.36, and 1.80 95%SI=1.28, 2.54, respectively). Studies using non-neoplastic DNA had higher variance than those based on tumor-infiltrated tissue DNA, half reported implausibly high EE, and many were susceptible to design and analysis errors that would bias estimates of association away from the null. Conclusions: We found little relative bias in the summary estimates of association, either overall or when limited to the tumor-infiltrated tissue DNA studies. Three guideline panels, based on robust evidence, recommend against CYP2D6 genotype-guided tamoxifen therapy. Alternatives for optimizing the effectiveness of tamoxifen therapy, such as assuring adherence and persistence, are more likely to achieve clinically important benefits. Citation Format: Ahern TP, Hertz DL, Damkier P, Ejlertsen B, Hamilton-Dutoit SJ, Rae JM, Regan MM, Thompson AM, Lash TL, Cronin-Fenton DP. CYP2D6 genotype and breast cancer recurrence in tamoxifen treated patients: An evaluation of the importance of loss-of-heterozygosity. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-23.

Aim The Pringle maneuver is a way to reduce blood loss during liver surgery. However, this may result in ischemia/reperfusion injury in the development of which Kupffer cells play a central role. Corticosteroids are known to have anti-inflammatory effects. Our aim was to investigate whether a conjugate of dexamethasone and antibody against the CD163 macrophage cell surface receptor could reduce ischemia/reperfusion injury in the rat liver. Methods Thirty-six male Wistar rats were used for the experiments. Animals were randomly divided into four groups of eight receiving anti-CD163-dexamethasone, high dose dexamethasone, low dose dexamethasone or placebo intravenously 18 h before laparotomy with subsequent 60 min of liver ischemia. After reperfusion for 24 h the animals had their liver removed. Bloods were drawn 30 min and 24 h post ischemia induction. Liver cell apoptosis and necrosis were analyzed by stereological quantification. Results After 24 h' reperfusion, the fraction of cell in non-necrotic tissues exhibiting apoptotic profiles was significantly lower in the high dose dexamethasone (p = 0.03) and anti-CD163-dex (p = 0.03) groups compared with the low dose dexamethasone and placebo groups. There was no difference in necrotic cell volume between groups. After 30 min of reperfusion, levels of haptoglobin were significantly higher in the anti-CD163-dex and high dose dexamethasone groups. Alanine aminotransferase and alkaline phosphatase were significantly higher in the high dose dexamethasone group compared to controls after 24 h' reperfusion. Conclusions We show that pharmacological preconditioning with anti-CD163-dex and high dose dexamethasone reduces the number of apoptotic cells following ischemia/reperfusion injury., Highlights • We investigated the effect of pharmacologic preconditioning with HDD, LDD and anti-CD163-dex on ischemia/reperfusion injury. • Liver cell apoptosis and necrosis were analyzed by stereological quantification. • Anti-CD163-dex and high dose dexamethasone reduces the number of apoptotic cells following ischemia/reperfusion injury.

AIM To evaluate the liver regeneration capacity (LRC) after partial hepatectomy (PH) in experimental non-alcoholic steatohepatitis (NASH). METHODS Fifty-four female rats were fed a high-fat, high-cholesterol diet (HFCD, 65% fat, 1% cholesterol) or standard diet (STD) for 16 wk. A 70% PH was performed and the animals were euthanised before PH or 2 or 5 d post- PH. LRC was evaluated using: The total number of Ki-67 positive hepatocytes in the caudate lobe, N(Ki-67, lobe) evaluated in a stereology-based design, the regenerated protein ratio (RPR), prothrombin-proconvertin ratio (PP), and mRNA expression of genes related to regeneration. RESULTS The HFCD NASH model showed significant steatosis with ballooning and inflammation, while no fibrosis was present. Mortality was similar in HFCD and STD animals following PH. HFCD groups were compared to respective STD groups and HFCD animals had a significantly elevated alanine transaminase at baseline (P < 0.001), as well as a significantly elevated bilirubin at day 2 after PH (P < 0.05). HFCD animals had a higher N(Ki-67, lobe) at baseline, (P < 0.0001), day 2 after PH (P = 0.06) and day 5 after PH (P < 0.025). We found no significant difference in RPR or PP neither 2 or 5 d post-PH. Expression of liver regeneration genes (e.g., hepatic growth factor) was higher at both day 2 and 5 post-PH in HFCD groups (P < 0.05). CONCLUSION NASH rats had a preserved LRC after hepatectomy when compared to STD rats. The methods and models of NASH are essential in understanding and evaluating LRC.

BACKGROUND AND AIMS: Hepatic cholesterol deposition drives inflammation and fibrosis in non-alcoholic steatohepatitis (NASH). The Niemann-Pick type C2 (NPC2) protein plays an important role in regulating intracellular cholesterol trafficking and homeostasis. We hypothesized that intravenous NPC2 supplementation reduces cholesterol accumulation, hepatic inflammation and fibrogenesis in a nutritional NASH rat model.METHODS: Rats were fed a high-fat, high-cholesterol (HFHC) diet for four weeks resulting in moderately severe NASH. Animals were treated with intravenous NPC2 or placebo twice weekly for either the last two weeks or the entire four weeks. End-points were liver/body- and spleen/body weight ratios, histopathological NASH scores, fibrosis, serum liver enzymes, cholesterol, lipoproteins, cytokines, and quantitative polymerase chain reaction derived hepatic gene expression related to cholesterol metabolism, inflammation, and fibrosis.RESULTS: HFHC rats developed hepatomegaly, non-fibrotic NASH histopathology, elevated liver enzymes, serum cholesterol, and pro-inflammatory cytokines. Their sterol regulatory element binding factor 2 (SREBF2) and low-density lipoprotein receptor (LDL-R) mRNAs were down-regulated compared with rats on standard chow. NPC2 did not improve liver weight, histopathology, levels of serum liver enzymes or pro-inflammatory tumor necrosis factor-α (TNFα), Interleukin (IL)-6, or IL-1β in HFHC rats. Two weeks of NPC2 treatment lowered hepatic TNFα and COL1A1 mRNA expression. However, this effect was ultimately reversed following additional two weeks of treatment. Four weeks NPC2 treatment of rats raised ATP-binding cassette A1 (ABCA1) and low-density lipoprotein receptor (LDLR) mRNAs in the liver, concurrent with a strong tendency towards higher serum high-density lipoprotein (HDL). Furthermore, the peroxisome proliferator activated receptor-ɣ (PPARG) gene expression was reduced.CONCLUSIONS: NPC2 proved inefficient at modifying robust hepatic NASH end-points in a HFHC NASH model. Nonetheless, our data suggest that hepatic ABCA1 expression and reverse cholesterol transport were upregulated by NPC2 treatment, thus presenting putative therapeutic effects in diseases associated with deregulated lipid metabolism.

Background & Aims: In non-alcoholic steatohepatitis (NASH), the function of urea cycle enzymes (UCEs) may be affected, resulting in hyperammonemia and the risk of disease progression. We aimed to determine whether the expression and function of UCEs are altered in an animal model of NASH and in patients with non-alcoholic fatty liver disease (NAFLD), and whether this process is reversible. Methods: Rats were first fed a high-fat, high-cholesterol diet for 10 months to induce NASH, before being switched onto a normal chow diet to recover. In humans, we obtained liver biopsies from 20 patients with steatosis and 15 with NASH. Primary rat hepatocytes were isolated and cultured with free fatty acids. We measured the gene and protein expression of ornithine transcarbamylase (OTC) and carbamoylphosphate synthetase (CPS1), as well as OTC activity, and ammonia concentrations. Moreover, we assessed the promoter methylation status of OTC and CPS1 in rats, humans and steatotic hepatocytes. Results: In NASH animals, gene and protein expression of OTC and CPS1, and the activity of OTC, were reversibly reduced. Hypermethylation of Otc promoter genes was also observed. Additionally, in patients with NAFLD, OTC enzyme concentration and activity were reduced and ammonia concentrations were increased, which was further exacerbated in those with NASH. Furthermore, OTC and CPS1 promoter regions were hypermethylated. In primary hepatocytes, induction of steatosis was associated with Otc promoter hypermethylation, a reduction in the gene expression of Otc and Cps1, and an increase in ammonia concentration in the supernatant. Conclusion: NASH is associated with a reduction in the gene and protein expression, and activity, of UCEs. This results in hyperammonemia, possibly through hypermethylation of UCE genes and impairment of urea synthesis. Our investigations are the first to describe a link between NASH, the function of UCEs, and hyperammonemia, providing a novel therapeutic target. Lay summary: In patients with fatty liver disease, the enzymes that convert nitrogen waste into urea may be affected, leading to the accumulation of ammonia, which is toxic. This accumulation of ammonia can lead to scar tissue development, increasing the risk of disease progression. In this study, we show that fat accumulation in the liver produces a reversible reduction in the function of the enzymes that are involved in detoxification of ammonia. These data provide potential new targets for the treatment of fatty liver disease. (C) 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Since coffee may help to prevent the development of metabolic syndrome (MetS), we aimed to evaluate the short- and long-term effects of a coffee-based supplement on different features of diet-induced MetS. In this study, 24 Sprague Dawley rats were divided into control or nutraceuticals groups to receive a high-fat/high-fructose diet with or without a mixture of caffeic acid (30 mg/day), trigonelline (20 mg/day), and cafestol (1 mg/day) for 12 weeks. An additional 11 rats were assigned to an acute crossover study. In the chronic experiment, nutraceuticals did not alter body weight or glycemic control, but improved fed hyperinsulinemia (mean difference = 30.80 mU/L, p = 0.044) and homeostatic model assessment-insulin resistance (HOMA-IR) (mean difference = 15.29, p = 0.033), and plasma adiponectin levels (mean difference = -0.99 µg/mL, p = 0.048). The impact of nutraceuticals on post-prandial glycemia tended to be more pronounced after acute administration than at the end of the chronic study. Circulating (mean difference = 4.75 U/L, p = 0.014) and intrahepatocellular alanine transaminase activity was assessed by hyperpolarized-13C nuclear magnetic resonance NMR spectroscopy and found to be reduced by coffee nutraceuticals at endpoint. There was also a tendency towards lower liver triglyceride content and histological steatosis score in the intervention group. In conclusion, a mixture of coffee nutraceuticals improved insulin sensitivity and exhibited hepatoprotective effects in a rat model of MetS. Higher dosages with or without caffeine deserve to be studied in the future.

Background: Expression of human paracrine hormones stanniocalcin 1 (STC1) and stanniocalcin 2 (STC2) may potentiate late breast cancer recurrence. We tested the hypothesis that expression of STC1 and STC2 in primary breast tumors is more strongly associated with late versus early recurrences. Methods: A total of 541 estrogen receptor–positive, tamoxifen-treated (ER+/TAM+) and 300 ER-negative, tamoxifen-untreated (ER−/TAM−) breast cancer patients who experienced recurrence within 10 years of primary diagnosis and matched recurrence-free controls were selected from a cohort of 11,251 Danish breast cancer patients diagnosed with stage I, II, or III breast cancer during 1985 to 2001. The association between IHC expression of STC1 and STC2 in primary breast tumor tissue microarrays and breast cancer recurrence was evaluated within median time to recurrence quintiles. Results: The association between STC1 expression, dichotomized as positive or negative, and recurrence was strongly positive for the final time quintile (6–10 years postdiagnosis) in the ER+/TAM+ group [aOR = 2.70; 95% confidence interval (CI): 1.22–5.98]. Regression of the log ORs relating dichotomous STC1 and STC2 expression to recurrence by median time to recurrence (year) resulted in a relatively large positive effect estimate for STC1 (β = 0.16; 95% CI, −0.03–0.36) and a near-null positive effect estimate for STC2 (β = 0.04; 95% CI, −0.14–0.21). Conclusions: Our results suggest a stronger association between primary tumor STC1 expression and late recurrence, as opposed to early recurrence, although no clear trend was apparent. Impact: STC1 expression in the primary tumor may potentiate late recurrences, suggesting dormancy pathways that merit further investigation. Cancer Epidemiol Biomarkers Prev; 27(6); 653–9. ©2018 AACR.

We describe the application of proteomic techniques for protein profiling and biomarker discovery in malignant lymphoma. Hematologic malignancies are primarily characterized by their clinical, morphological, immunophenotypical, and molecular-genetic features. However, when based on these parameters, apparently identical lymphomas may show distinct clinical courses, suggesting underlying biological heterogeneity. Recent proteomic analyses have identified differences in protein expression both with regard to subclassification of the malignant lymphoma entities, as well as in correlation with clinical outcome. In this review, studies on quantification of differential protein expression in and between malignant lymphoma entities are included. Studies are included that are based on patient samples, that is, serum/plasma or cytological specimens, as well as intact tumor tissues, together with studies that focus on tumor cells alone, or in conjunction with the tumor microenvironment. For biomarker discovery in malignant lymphoma, these approaches are used to uncover the underlying biological mechanisms and identify proteins with potential diagnostic and prognostic utility, either as predictive biomarkers or as novel future treatment targets.

Post-transplant lymphoproliferative disorders (PTLDs) are potentially fatal, often Epstein-Barr virus (EBV)-driven neoplasias developing in immunocompromised hosts. Initial treatment usually consists of a reduction in immunosuppressive therapy and/or rituximab with or without chemotherapy. However, patients who relapse do poorly, and new treatment options are warranted. With the introduction of the immunoconjugate brentuximab vedotin, the CD30 antigen has become an effectively targetable molecule. Therefore, we investigated the frequency and level of CD30 expression in PTLDs. We identified 108 patients with PTLDs diagnosed during 1994-2011, of whom 62 had adequate paraffin-embedded tissue for tissue microarray construction. Immunohistochemical expression of CD30 was consistently detected in all types of PTLD (overall 85.25%), including the monomorphic subtypes, and was correlated with a more favorable outcome. For diffuse large B-cell lymphoma (DLBCL)-type PTLD this was regardless of EBV status, and remained significant in multivariate analysis. Cell-of-origin had no independent prognostic value in our series of DLBCL PTLD.

OBJECTIVES: Inflammatory activation of resident hepatic macrophages (Kupffer cells) by portal-derived lipopolysaccharide (LPS) has a primary role in animal models of alcoholic liver disease, but it has not been systematically or longitudinally studied in human alcoholic hepatitis (AH).METHODS: We followed 50 patients with AH for 30 days. 26 patients with stable alcoholic cirrhosis and 20 healthy individuals were controls. We measured the plasma (P) concentrations of soluble CD163 (sCD163; a specific marker of inflammatory macrophage activation) and the expression of CD163 in liver tissue by immunohistochemistry and stereology of liver biopsies. We also measured the key components of the LPS pathway, P-LPS, sCD14, and LPS-binding protein (LBP), by enzyme-linked immunosorbent assay (ELISA). The 84-day mortality was registered.RESULTS: At study entry, the sCD163 concentration was 10-fold higher than in the healthy controls and 30% higher than in the stable cirrhotics (P0.35, PCONCLUSIONS: The hepatic inflammation of human AH involves marked activation of hepatic macrophages, likely via the LPS pathway. Hepatic macrophages may thus present a target for biological therapy of AH.

BACKGROUND: Validation studies of the Danish Breast Cancer Group (DBCG) registry show good agreement with medical records for adjuvant treatment data, but inconsistent recurrence information. No studies have validated changes in menopausal status or endocrine therapy during follow-up. In a longitudinal study, we validated DBCG data using medical records as the gold standard.MATERIAL AND METHODS: From a cohort of 5959 premenopausal women diagnosed during 2002-2010 with stage I-III breast cancer, we selected 151 patients - 77 estrogen-receptor-positive and 74 estrogen-receptor-negative - from three hospitals. We assessed the validity of DBCG registry data on patient, tumor, and treatment factors, and follow-up information on menopausal transition, changes in endocrine therapy, and recurrence. We computed positive predictive values (PPVs) with 95% confidence intervals (95%CI).RESULTS: Agreement was near perfect for tumor size, lymph node involvement, receptor status, surgery type, and receipt of radiotherapy, chemotherapy, or tamoxifen treatment. The PPV for a change in endocrine therapy in the DBCG was 96% (95%CI = 83, 100). The PPV for menopausal transition was 61% (95%CI = 42, 77). The PPV for DBCG-recorded recurrence was 100%. However, of 19 patients who had a recurrence documented in their medical record, 13 had the recurrence registered in DBCG.CONCLUSIONS: DBCG data are valid for most epidemiological studies of breast cancer treatment. Data on menopausal transition may be less valid, though this interpretation depends on the suitability of medical records for making this assessment. Although recurrence is missing for some, this would not bias most ratio measures of association.

BACKGROUND: Long-term excess alcohol intake predisposes to infectious diseases. The hepatic acute phase response is a component of the innate immune system and is part of the first line of defense against invading pathogens, which may be compromised by alcohol. We aimed to investigate whether an induced acute phase response is impaired in long-term ethanol-fed rats.METHODS: For six weeks, rats were either fed a Lieber-DeCarli ethanol-containing (36% as calories) liquid diet ad libitum or calorically pair-fed. Then, the rats were injected intraperitoneally with a low-dose of lipopolysaccharide (LPS) (0.5 mg/kg) to induce an acute phase response. Two hours after LPS, we measured the plasma concentrations of an array of inflammatory cytokines. Twenty-four hours after LPS, we measured the hepatic mRNA expression and serum concentrations of prominent rat acute phase proteins.RESULTS: Ethanol-fed rats showed either no liver histopathological changes or varying degrees of steatosis. Ethanol feeding decreased the spontaneous liver mRNA expression of the prevailing acute phase protein alpha-2-macroglobulin by 30% (PCONCLUSIONS: Long-term ethanol exposure in rats reduces the spontaneous hepatic mRNA expression of alpha-2-macroglobulin and markedly impairs the hepatic acute phase response to endotoxin, despite higher pro-inflammatory cytokine release. The same phenomenon may contribute to the increased susceptibility to infections observed in humans with long-term excess alcohol intake. This article is protected by copyright. All rights reserved.

Clinical trials (CTs) are needed to improve the outcome for peripheral T-cell lymphomas (PTCL), and accrual into CTs is one of the main recommendations in international treatment guidelines. The use of risk-adapted strategies has been suggested as a way to optimize treatment outcome in PTCL. The aim of the present study was to evaluate CT eligibility and selected prognostic indices in a population-based PTCL cohort of 481 PTCL patients identified from the Danish Lymphoma Registry in the period 2000–2010. According to five predefined parameters (age, performance status, P-creatinine, P-ALAT and measurable tumour lesion), patients were subdivided into four groups: ‘younger fit’, ‘elderly fit’, ‘frail’ and ‘not CT eligible’. International prognostic index (IPI), prognostic index for T-cell lymphoma (PIT) and anaplastic lymphoma kinase (ALK) protein expression were tested at subtype-specific level. Overall, 41% of the patients were considered eligible for interventional CTs implicating curatively intended multiagent chemotherapy, including, if considered appropriate, consolidating stem cell transplantation (SCT), as part of the upfront management strategy. Moreover, 28% was elderly fit and eligible for interventional CT, including those with SCT as part of the trial design. Approximately 7% were defined as ‘too frail’ for aggressive treatment schedules, whereas 24% were deemed not to be eligible for any CT. Both overall and progression-free survivals were effectively predicted by IPI and PIT (p

PURPOSE: Overexpression of 14-3-3ζ has been linked to breast cancer recurrence in several studies, including studies assessing its effect on tamoxifen resistance. The study was performed to estimate the effect of 14-3-3ζ and differentiate potential prognostic or predictive utility.METHODS: A case-control study, nested in a population of 11,251 females residing on the Jutland Peninsula of Denmark, was performed. Participants were aged 35-69, diagnosed with stage I, II, or III breast cancer between 1985 and 2001, and registered with the Danish Breast Cancer Cooperative Group. We identified 541 recurrent breast cancer cases with estrogen receptor-positive disease treated with tamoxifen for at least 1 year (ER+/TAM+) and 300 cases with estrogen receptor-negative disease never treated with tamoxifen (ER-/TAM-). We matched cases to controls on ER/TAM status, date of surgery, menopausal status, stage, and county. 14-3-3ζ expression was assessed using immunohistochemistry on tissue microarrays. We computed the odds ratio (OR) associating 14-3-3ζ expression with breast cancer recurrence adjusting for confounding using logistic regression. A quantitative bias analysis was performed to account for bias due to expression assay methods.RESULTS: Associations for cytoplasmic and nuclear 14-3-3ζ staining above the 50th percentile were near null in both ER+/TAM+ and ER-/TAM- patients. When examining combined 14-3-3ζ staining, the association increased in the ER+/TAM+ group (adjusted OR 1.44, 95% confidence interval (CI) 1.05, 1.99). A nearly twofold increase in odds of recurrence was observed in above the 75th percentile staining of combined 14-3-3ζ, both for ER+/TAM+ patients (adjusted OR 1.93, 95% CI 1.15, 3.24) and ER-/TAM- patients (adjusted OR 1.93, 95% CI 1.03, 3.62), indicating potential prognostic utility.CONCLUSION: Evidence is lacking to conclude that 14-3-3ζ is a useful marker of tamoxifen resistance; however, 14-3-3ζ expression is a potentially useful prognostic marker of breast cancer recurrence. Independent utility beyond established prognostic markers needs to be determined.

SummaryBackground Cadherin switch in melanoma, with loss of E-cadherin and upregulation of N-cadherin, is believed to underlie melanoma cell detachment from the epidermis and promotion of dermal and vascular melanoma invasion. The tumour suppressor phosphatase and tensin homolog (PTEN) has been suggested as a potential regulator of this cadherin switch. Objectives To study the biological and clinical implications of cadherin switch and PTEN expression in melanoma progression. Methods We constructed tissue microarrays from primary tumour samples from 394 formalin-fixed paraffin-embedded melanomas diagnosed between 2001 and 2006. Median follow-up was 10 years. Tissue microarray sections were stained by immunohistochemistry for E-cadherin, N-cadherin and PTEN, and expression was analysed semiquantitatively. Results Breslow thickness correlated strongly with reduced/absent PTEN expression (P

Background Ischemic preconditioning (IPC) has been shown to protect the liver against ischemia-reperfusion (I/R) injuries. However, ischemic post-conditioning has received little attention. The aim of the present study was to quantify and compare the hepato-protective properties of IPC and IPO, for the first time, using unbiased design-based stereological methods. Methods We divided 67 rats into four groups: sham, liver ischemia (LI), IPC, and IPO. Rats were subjected to 60 min LI, followed by 4- or 24-h reperfusion. We performed quantification of (NVR) and apoptotic cell profile number. Results We observed no significant differences in NVR between ischemic groups after 4 h. After 24-h reperfusion, NVR had increased to 70% in the LI group, compared with 51% ( P = 0.02) and 49% ( P = 0.01) in the IPC and IPO groups, respectively. After 4-h reperfusion, the apoptotic cell number was significantly higher in all ischemic groups than in the sham group; we detected no difference between ischemic groups. After 24-h reperfusion, we detected a significantly lower number of apoptotic cell profiles in the IPC group than in the LI group ( P = 0.02). The mean number of apoptotic cell profiles decreased insignificantly in the IPO group ( P = 0.06). Liver parameters were at all time comparable between groups. Conclusions After I/R, IPC and IPO reduce the degree of hepatocellular injury. Both methods are equally efficient at preventing hepatocellular necrosis. Furthermore, apoptosis is significantly lower after IPC.

This case describes the clinical course and treatment of a 17-year-old male patient with advanced hepatocellular carcinoma (HCC) arising in a non-cirrhotic liver. The disease was thought to be caused by a congenital cholestatic syndrome associated with intermittent oedema in childhood, resembling the rare Aagenaes syndrome. Treatment choices in advanced HCC arising in adolescence are discussed.