Intraductal papillary neoplasms of the bile duct: stepwise progression to carcinoma involves common molecular pathways

Intraductal papillary neoplasms of the bile duct are still poorly characterized regarding (1) their molecular alterations during the development to invasive carcinomas, (2) their subtype stratification and (3) their biological behavior. We performed a multicenter study that analyzed these issues in a large European cohort. Intraductal papillary neoplasms of the bile duct from 45 patients were graded and subtyped using mucin markers and CDX2. In addition, tumors were analyzed for common oncogenic pathways, and the findings were correlated with subtype and grade. Data were compared with those from 22 extra- and intrahepatic cholangiocarcinomas. Intraductal papillary neoplasms showed a development from preinvasive low- to high-grade intraepithelial neoplasia to invasive carcinoma. Molecular and immunohistochemical analysis revealed mutated KRAS, overexpression of TP53 and loss of p16 in low-grade intraepithelial neoplasia, whereas loss of SMAD4 was found in late phases of tumor development. Alterations of HER2, EGFR, β-catenin and GNAS were rare events. Among the subtypes, pancreato-biliary (36%) and intestinal (29%) were the most common, followed by gastric (18%) and oncocytic (13%) subtypes. Patients with intraductal papillary neoplasm of the bile duct showed a slightly better overall survival than patients with cholangiocarcinoma (hazard ratio (cholangiocarcinoma versus intraductal papillary neoplasm of the bile duct): 1.40; 95% confidence interval: 0.46-4.30; P=0.552). The development of biliary intraductal papillary neoplasms of the bile duct follows an adenoma-carcinoma sequence that correlates with the stepwise activation of common oncogenic pathways. Further large trials are needed to investigate and verify the finding of a better prognosis of intraductal papillary neoplasms compared with conventional cholangiocarcinoma.

Oxygen regulates molecular mechanisms of cancer progression and metastasis

Oxygen is the basic molecule which supports life and it truly is “god's gift to life.” Despite its immense importance, research on “oxygen biology” has never received the light of the day and has been limited to physiological and biochemical studies. It seems that in modern day biology, oxygen research is summarized in one word “hypoxia.” Scientists have focused on hypoxia-induced transcriptomics and molecular–cellular alterations exclusively in disease models. Interestingly, the potential of oxygen to control the basic principles of biology like homeostatic maintenance, transcription, replication, and protein folding among many others, at the molecular level, has been completely ignored. Here, we present a perspective on the crucial role played by oxygen in regulation of basic biological phenomena. Our conclusion highlights the importance of establishing novel research areas like oxygen biology, as there is great potential in this field for basic science discoveries and clinical benefits to the society.

Hierarchical accumulation of RyR post-translational modifications drives disease progression in dystrophic cardiomyopathy

AIMS:Duchenne muscular dystrophy (DMD) is a muscle disease with serious cardiac complications. Changes in Ca(2+) homeostasis and oxidative stress were recently associated with cardiac deterioration, but the cellular pathophysiological mechanisms remain elusive. We investigated whether the activity of ryanodine receptor (RyR) Ca(2+) release channels is affected, whether changes in function are cause or consequence and which post-translational modifications drive disease progression. METHODS AND RESULTS:Electrophysiological, imaging, and biochemical techniques were used to study RyRs in cardiomyocytes from mdx mice, an animal model of DMD. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. Nevertheless, myocytes from mdx pups exhibited exaggerated Ca(2+) responses to mechanical stress and 'hypersensitive' excitation-contraction coupling, hallmarks of increased RyR Ca(2+) sensitivity. Both were normalized by antioxidants, inhibitors of NAD(P)H oxidase and CaMKII, but not by NO synthases and PKA antagonists. Sarcoplasmic reticulum Ca(2+) load and leak were unchanged in young mdx mice. However, by the age of 4-5 months and in senescence, leak was increased and load was reduced, indicating disease progression. By this age, all pharmacological interventions listed above normalized Ca(2+) signals and corrected changes in ECC, Ca(2+) load, and leak. CONCLUSION:Our findings suggest that increased RyR Ca(2+) sensitivity precedes and presumably drives the progression of dystrophic cardiomyopathy, with oxidative stress initiating its development. RyR oxidation followed by phosphorylation, first by CaMKII and later by PKA, synergistically contributes to cardiac deterioration.

Multidrug resistance protein 4 (MRP4) expression in prostate cancer is associated with androgen signaling and decreases with tumor progression

Multidrug resistance protein 4 (MRP4) is a transmembrane transport protein found in many cell types and is involved in substrate-specific transport of endogenous and exogenous substrates. Recently, it has shown to be expressed in prostate cancer cell lines and to be among the most commonly upregulated transcripts in prostate cancer, although a comprehensive expression analysis is lacking so far. We aimed to investigate its expression by immunohistochemistry in a larger cohort of neoplastic and nonneoplastic prostate tissues (n = 441) and to correlate its expression with clinicopathological parameters including PSA-free survival times and molecular correlates of androgen signaling (androgen receptor (AR), prostate-specific antigen (PSA), and forkhead box A (FoxA)). MRP4 is widely expressed in benign and neoplastic prostate epithelia, but its expression gradually decreases during tumor progression towards castrate-resistant disease. Concordantly, it correlated with conventional prognosticators of disease progression and-within the group of androgen-dependent tumors-with AR and FoxA expression. Moreover, lower levels of MRP4 expression were associated with shorter PSA relapse-free survival times in the androgen-dependent group. In benign tissues, we found zone-dependent differences of MRP4 expression, with the highest levels in the peripheral and central zones. Although MRP4 is known to be regulated in prostate cancer, this study is the first to demonstrate a gradual downregulation of MRP4 protein during malignant tumor progression and a prognostic value of this loss of expression.

Trastuzumab treatment beyond progression in advanced breast cancer: patterns of care in six Swiss breast cancer centers.

BACKGROUND Trastuzumab is an established treatment for HER2-positive breast cancer (BC). We analyzed Swiss patterns of care in patients with HER2-positive BC after disease progression on trastuzumab-containing therapy for metastatic BC (MBC). PATIENTS AND METHODS A retrospective analysis was performed in six Swiss BC centers. Patients with HER2-positive MBC treated with at least one infusion of trastuzumab for advanced disease between January 2006 and December 2007 were identified. Treatment patterns in first and further lines were analyzed. RESULTS All of the 72 identified patients received trastuzumab as their first palliative anti-HER2 therapy, either as monotherapy (n = 23) or in combination with chemotherapy (typically taxane or vinorelbine; n = 49). Median time to progression was 8.1, 8.0 and 7.9 months in the monotherapy, trastuzumab-taxane and trastuzumab-vinorelbine cohorts, respectively. After progression on first-line anti-HER2 therapy, trastuzumab was continued in 67 of 68 patients who received further therapy. One patient received second-line lapatinib plus capecitabine. The median duration of anti-HER2 therapy was 20 months. Patients received a median of 4 lines of anti-HER2 therapy. CONCLUSIONS Durable responses were achieved with repeated exposure to anti-HER2 therapy. In a selected patient population, trastuzumab monotherapy appears to be a reasonable first-line treatment option.

Highly pathogenic adapted HIV-1 strains limit host immunity and dictate rapid disease progression.

OBJECTIVE: The study of HIV-1 rapid progressors has been limited to specific case reports. Nevertheless, identification and characterization of the viral and host factors involved in rapid progression are crucial when attempting to uncover the correlates of rapid disease outcome. DESIGN: We carried out comparative functional analyses in rapid progressors (n = 46) and standard progressors (n = 46) early after HIV-1 seroconversion (≤1 year). The viral traits tested were viral replicative capacity, co-receptor usage, and genomic variation. Host CD8 T-cell responses, humoral activity, and HLA immunogenetic markers were also determined. RESULTS: Our data demonstrate an unusual convergence of highly pathogenic HIV-1 strains in rapid progressors. Compared with standard progressors, rapid progressor viral strains show higher in-vitro replicative capacity (81.5 vs. 67.9%; P = 0.025) and greater X4/DM co-receptor usage (26.3 vs. 2.8%; P = 0.006) in early infection. Limited or absent functional HIV-1 CD8 T-cell responses and neutralizing activity were measured in rapid progressors. Moreover, the increase in common HLA allele-restricted CD8 T-cell escape mutations in rapid progressors acts as a signature of uncontrolled HIV-1 replication and early impairment of adaptive cellular responses. CONCLUSION: Our data support a dominant role for viral factors in rapid progressors. Robust HIV-1 replication and intrinsic viral properties limit host adaptive immune responses, thus driving rapid disease progression.

Biokinetics of nanoparticles and susceptibility to particulate exposure in a murine model of cystic fibrosis.

BACKGROUND Persons with cystic fibrosis (CF) are at-risk for health effects from ambient air pollution but little is known about the interaction of nanoparticles (NP) with CF lungs. Here we study the distribution of inhaled NP in a murine CF model and aim to reveal mechanisms contributing to adverse effects of inhaled particles in susceptible populations. METHODS Chloride channel defective CftrTgH (neoim) Hgu mice were used to analyze lung function, lung distribution and whole body biokinetics of inhaled NP, and inflammatory responses after intratracheal administration of NP. Distribution of 20-nm titanium dioxide NP in lungs was assessed on ultrathin sections immediately and 24 h after a one-hour NP inhalation. NP biokinetics was deduced from total and regional lung deposition and from whole body translocation of inhaled 30-nm iridium NP within 24 h after aerosol inhalation. Inflammatory responses were assessed within 7 days after carbon NP instillation. RESULTS Cftr mutant females had moderately reduced lung compliance and slightly increased airway resistance compared to wild type mice. We found no genotype dependent differences in total, regional and head deposition or in secondary-organ translocation of inhaled iridium NP. Titanium dioxide inhalation resulted in higher NP uptake by alveolar epithelial cells in Cftr mutants. Instillation of carbon NP induced a comparable acute and transient inflammatory response in both genotypes. The twofold increase of bronchoalveolar lavage (BAL) neutrophils in Cftr mutant compared to wild type mice at day 3 but not at days 1 and 7, indicated an impaired capacity in inflammation resolution in Cftr mutants. Concomitant to the delayed decline of neutrophils, BAL granulocyte-colony stimulating factor was augmented in Cftr mutant mice. Anti-inflammatory 15-hydroxyeicosatetraenoic acid was generally significantly lower in BAL of Cftr mutant than in wild type mice. CONCLUSIONS Despite lacking alterations in lung deposition and biokinetics of inhaled NP, and absence of significant differences in lung function, higher uptake of NP by alveolar epithelial cells and prolonged, acute inflammatory responses to NP exposure indicate a moderately increased susceptibility of lungs to adverse effects of inhaled NP in Cftr mutant mice and provides potential mechanisms for the increased susceptibility of CF patients to air pollution.

The craniosacral progression of muscle development influences the emergence of neuromuscular junction alterations in a severe murine model for spinal muscular atrophy.

AIMS As 4-day-old mice of the severe spinal muscular atrophy (SMA) model (dying at 5-8 days) display pronounced neuromuscular changes in the diaphragm but not the soleus muscle, we wanted to gain more insight into the relationship between muscle development and the emergence of pathological changes and additionally to analyse intercostal muscles which are affected in human SMA. METHODS Structures of muscle fibres and neuromuscular junctions (NMJs) of the diaphragm, intercostal and calf muscles of prenatal (E21) and postnatal (P0 and P4) healthy and SMA mice were analysed by light and transmission electron microscopy. NMJ innervation was studied by whole mount immunofluorescence in diaphragms of P4 mice. RESULTS During this period, the investigated muscles still show a significant neck-to-tail developmental gradient. The diaphragm and calf muscles are most and least advanced, respectively, with respect to muscle fibre fusion and differentiation. The number and depth of subsynaptic folds increases, and perisynaptic Schwann cells (PSCs) acquire a basal lamina on their outer surface. Subsynaptic folds are connected to an extensive network of tubules and beaded caveolae, reminiscent of the T system in adult muscle. Interestingly, intercostal muscles from P4 SMA mice show weaker pathological involvement (that is, vacuolization of PSCs and perineurial cells) than those previously described by us for the diaphragm, whereas calf muscles show no pathological changes. CONCLUSION SMA-related alterations appear to occur only when the muscles have reached a certain developmental maturity. Moreover, glial cells, in particular PSCs, play an important role in SMA pathogenesis.

Correlation of serum and urinary matrix metalloproteases/tissue inhibitors of metalloproteases with subclinical allograft fibrosis in renal transplantation

Progressive interstitial fibrosis and tubular atrophy (IF/TA) is a leading cause of chronic allograft dysfunction. Increased extracellular matrix remodeling regulated by matrix metalloproteases (MMPs) and their inhibitors (TIMPs) has been implicated in the development of IF/TA. The aim of this study was to investigate whether urinary/serum MMPs/TIMPs correlate with subclinical IF/TA detected in surveillance biopsies within the first 6months post-transplant. We measured eight different MMPs/TIMPs simultaneously in urine and serum samples from patients classified as normal histology (n=15), IF/TA 1 (n=15) and IF/TA 2-3 (n=10). There was no difference in urinary MMPs/TIMPs among the three groups, and only 1/8 serum MMPs/TIMPs (i.e. MMP-1) was significantly elevated in biopsies with IF/TA 2-3 (p=0.01). In addition, urinary/serum MMPs/TIMPs were not different between surveillance biopsies demonstrating an early development of IF/TA (i.e. delta IF/TA≥1 compared to a previous biopsy obtained three months before; n=11) and stable grade of IF/TA (i.e. delta IF/TA=0; n=20). Next, we investigated whether urinary/serum MMP/TIMP levels are elevated during acute subclinical tubulitis in surveillance biopsies obtained within the first 6months post-transplant (n=25). Compared to biopsies with normal histology, serum MMPs/TIMPs were not different; however, all urinary MMP/TIMP levels were numerically higher during subclinical tubulitis (MMP-1, MMP-7, TIMP-1 with p≤0.04). We conclude that urinary/serum MMPs/TIMPs do hardly correlate with existing or early developing IF/TA in surveillance biopsies obtained within the first 6months post-transplant. This could be explained by the dynamic process of extracellular matrix remodeling, which seems to be active during acute tubulo-interstitial injury/inflammation, but not in quiescent IF/TA.

Everolimus is a potent inhibitor of activated hepatic stellate cell functions in vitro and in vivo , while demonstrating anti-angiogenic activities

Progression of liver fibrosis to HCC (hepatocellular carcinoma) is a very complex process which involves several pathological phenomena, including hepatic stellate cell activation, inflammation, fibrosis and angiogenesis. Therefore inhibiting multiple pathological processes using a single drug can be an effective choice to curb the progression of HCC. In the present study, we used the mTOR inhibitor everolimus to observe its effect on the in vitro activation of hepatic stellate cells and angiogenesis. The results of the present study demonstrated that everolimus treatment blocked the functions of the immortalized human activated hepatic stellate cell line LX-2 without affecting the viability and migration of primary human stellate cells. We also observed that treatment with everolimus (20 nM) inhibited collagen production by activated stellate cells, as well as cell contraction. Everolimus treatment was also able to attenuate the activation of primary stellate cells to their activated form. Angiogenesis studies showed that everolimus blocked angiogenesis in a rat aortic ring assay and inhibited the tube formation and migration of liver sinusoidal endothelial cells. Finally, everolimus treatment reduced the load of tumoral myofibroblasts in a rat model of HCC. These data suggest that everolimus targets multiple mechanisms, making it a potent blocker of the progression of HCC from liver fibrosis.