Lesions hyper- to isointense to surrounding liver in the hepatobiliary phase of gadoxetic acid-enhanced MRI.

OBJECTIVES: Hyper- or isointensity in the hepatobiliary phase (HBP) of gadoxetic acid-enhanced MRI has high specificity for focal nodular hyperplasia (FNH) but may be present in hepatocellular adenoma and carcinoma (HCA/HCC). This study aimed to identify imaging characteristics differentiating FNH and HCA/HCC. MATERIALS AND METHODS: This multicenter retrospective cohort study included patients with pathology-proven FNH or HCA/HCC, hyper-/isointense in the HBP of gadoxetic acid-enhanced MRI between 2010 and 2020. Diagnostic performance of imaging characteristics for the differentiation between FNH and HCA/HCC were reported. Univariable analyses, multivariable logistic regression analyses, and classification and regression tree (CART) analyses were conducted. Sensitivity analyses evaluated imaging characteristics of B-catenin-activated HCA. RESULTS: In total, 124 patients (mean age 40 years, standard deviation 10 years, 108 female) with 128 hyper-/isointense lesions were included. Pathology diagnoses were FNH and HCA/HCC in 64 lesions (50%) and HCA/HCC in 64 lesions (50%). Imaging characteristics observed exclusively in HCA/HCC were raster and atoll fingerprint patterns in the HBP, sinusoidal dilatation on T2-w, hemosiderin, T1-w in-phase hyperintensity, venous washout, and nodule-in-nodule partification in the HBP and T2-w. Multivariable logistic regression and CART additionally found a T2-w scar indicating FNH, less than 50% fat, and a spherical contour indicating HCA/HCC. In our selected cohort, 14/48 (29%) of HCA were B-catenin activated, most (13/14) showed extensive hyper-/isointensity, and some had a T2-w scar (4/14, 29%). CONCLUSION: If the aforementioned characteristics typical for HCA/HCC are encountered in lesions extensively hyper- to isointense, further investigation may be warranted to exclude B-catenin-activated HCA. CLINICAL RELEVANCE: Hyper- or isointensity in the HBP of gadoxetic acid-enhanced MRI is specific for FNH, but HCA/HCC can also exhibit this feature. Therefore, we described imaging patterns to differentiate these entities. KEY POINTS: FNH and HCA/HCC have similar HBP intensities but have different malignant potentials. Six imaging patterns exclusive to HCA/HCC were identified in this lesion population. These features in liver lesions hyper- to isointense in the HBP warrant further evaluation.

How subjective CT image quality assessment becomes surprisingly reliable: pairwise comparisons instead of Likert scale.

OBJECTIVES: The aim of this study is to improve the reliability of subjective IQ assessment using a pairwise comparison (PC) method instead of a Likert scale method in abdominal CT scans. METHODS: Abdominal CT scans (single-center) were retrospectively selected between September 2019 and February 2020 in a prior study. Sample variance in IQ was obtained by adding artificial noise using dedicated reconstruction software, including reconstructions with filtered backprojection and varying iterative reconstruction strengths. Two datasets (each n = 50) were composed with either higher or lower IQ variation with the 25 original scans being part of both datasets. Using in-house developed software, six observers (five radiologists, one resident) rated both datasets via both the PC method (forcing observers to choose preferred scans out of pairs of scans resulting in a ranking) and a 5-point Likert scale. The PC method was optimized using a sorting algorithm to minimize necessary comparisons. The inter- and intraobserver agreements were assessed for both methods with the intraclass correlation coefficient (ICC). RESULTS: Twenty-five patients (mean age 61 years ± 15.5; 56% men) were evaluated. The ICC for interobserver agreement for the high-variation dataset increased from 0.665 (95%CI 0.396-0.814) to 0.785 (95%CI 0.676-0.867) when the PC method was used instead of a Likert scale. For the low-variation dataset, the ICC increased from 0.276 (95%CI 0.034-0.500) to 0.562 (95%CI 0.337-0.729). Intraobserver agreement increased for four out of six observers. CONCLUSION: The PC method is more reliable for subjective IQ assessment indicated by improved inter- and intraobserver agreement. CLINICAL RELEVANCE STATEMENT: This study shows that the pairwise comparison method is a more reliable method for subjective image quality assessment. Improved reliability is of key importance for optimization studies, validation of automatic image quality assessment algorithms, and training of AI algorithms. KEY POINTS: • Subjective assessment of diagnostic image quality via Likert scale has limited reliability. • A pairwise comparison method improves the inter- and intraobserver agreement. • The pairwise comparison method is more reliable for CT optimization studies.

Automated Assessment of T2-Weighted MRI to Differentiate Malignant and Benign Primary Solid Liver Lesions in Noncirrhotic Livers Using Radiomics.

RATIONALE AND OBJECTIVES: Distinguishing malignant from benign liver lesions based on magnetic resonance imaging (MRI) is an important but often challenging task, especially in noncirrhotic livers. We developed and externally validated a radiomics model to quantitatively assess T2-weighted MRI to distinguish the most common malignant and benign primary solid liver lesions in noncirrhotic livers. MATERIALS AND METHODS: Data sets were retrospectively collected from three tertiary referral centers (A, B, and C) between 2002 and 2018. Patients with malignant (hepatocellular carcinoma and intrahepatic cholangiocarcinoma) and benign (hepatocellular adenoma and focal nodular hyperplasia) lesions were included. A radiomics model based on T2-weighted MRI was developed in data set A using a combination of machine learning approaches. The model was internally evaluated on data set A through cross-validation, externally validated on data sets B and C, and compared to visual scoring of two experienced abdominal radiologists on data set C. RESULTS: The overall data set included 486 patients (A: 187, B: 98, and C: 201). The radiomics model had a mean area under the curve (AUC) of 0.78 upon internal validation on data set A and a similar AUC in external validation (B: 0.74 and C: 0.76). In data set C, the two radiologists showed moderate agreement (Cohen's κ: 0.61) and achieved AUCs of 0.86 and 0.82. CONCLUSION: Our T2-weighted MRI radiomics model shows potential for distinguishing malignant from benign primary solid liver lesions. External validation indicated that the model is generalizable despite substantial MRI acquisition protocol differences. Pending further optimization and generalization, this model may aid radiologists in improving the diagnostic workup of patients with liver lesions.

Generation of synthetic ground glass nodules using generative adversarial networks (GANs).

BACKGROUND: Data shortage is a common challenge in developing computer-aided diagnosis systems. We developed a generative adversarial network (GAN) model to generate synthetic lung lesions mimicking ground glass nodules (GGNs). METHODS: We used 216 computed tomography images with 340 GGNs from the Lung Image Database Consortium and Image Database Resource Initiative database. A GAN model retrieving information from the whole image and the GGN region was built. The generated samples were evaluated with visual Turing test performed by four experienced radiologists or pulmonologists. Radiomic features were compared between real and synthetic nodules. Performances were evaluated by area under the curve (AUC) at receiver operating characteristic analysis. In addition, we trained a classification model (ResNet) to investigate whether the synthetic GGNs can improve the performances algorithm and how performances changed as a function of labelled data used in training. RESULTS: Of 51 synthetic GGNs, 19 (37%) were classified as real by clinicians. Of 93 radiomic features, 58 (62.4%) showed no significant difference between synthetic and real GGNs (p ≥ 0.052). The discrimination performances of physicians (AUC 0.68) and radiomics (AUC 0.66) were similar, with no-significantly different (p = 0.23), but clinicians achieved a better accuracy (AUC 0.74) than radiomics (AUC 0.62) (p < 0.001). The classification model trained on datasets with synthetic data performed better than models without the addition of synthetic data. CONCLUSIONS: GAN has promising potential for generating GGNs. Through similar AUC, clinicians achieved better ability to diagnose whether the data is synthetic than radiomics.

A Prospective Multicenter Comparison Study of Risk-adapted Ultrasound-directed and Magnetic Resonance Imaging-directed Diagnostic Pathways for Suspected Prostate Cancer in Biopsy-naïve Men.

BACKGROUND: European Association of Urology guidelines recommend a risk-adjusted biopsy strategy for early detection of prostate cancer in biopsy-naïve men. It remains unclear which strategy is most effective. Therefore, we evaluated two risk assessment pathways commonly used in clinical practice. OBJECTIVE: To compare the diagnostic performance of a risk-based ultrasound (US)-directed pathway (Rotterdam Prostate Cancer Risk Calculator [RPCRC] #3; US volume assessment) and a magnetic resonance imaging (MRI)-directed pathway. DESIGN, SETTING, AND PARTICIPANTS: This was a prospective multicenter study (MR-PROPER) with 1:1 allocation among 21 centers (US arm in 11 centers, MRI arm in ten). Biopsy-naïve men with suspicion of prostate cancer (age ≥50 yr, prostate-specific antigen 3.0-50 ng/ml, ± abnormal digital rectal examination) were included. INTERVENTION: Biopsy-naïve men with elevated risk of prostate cancer, determined using RPCRC#3 in the US arm and Prostate Imaging Reporting and Data System scores of 3-5 in the MRI arm, underwent systematic biopsies (US arm) or targeted biopsies (MRI arm). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome was the proportion of men with grade group (GG) ≥2 cancer. Secondary outcomes were the proportions of biopsies avoided and GG 1 cancers detected. Categorical (nonparametric) data were assessed using the Mann-Whitney U test and χ2 tests. RESULTS AND LIMITATIONS: A total of 1965 men were included in the intention-to-treat population (US arm n = 950, MRI arm n = 1015). The US and MRI pathways detected GG ≥2 cancers equally well (235/950, 25% vs 239/1015, 24%; difference 1.2%, 95% confidence interval [CI] -2.6% to 5.0%; p = 0.5). The US pathway detected more GG 1 cancers than the MRI pathway (121/950, 13% vs 84/1015, 8.3%; difference 4.5%, 95% CI 1.8-7.2%; p < 0.01). The US pathway avoided fewer biopsies than the MRI pathway (403/950, 42% vs 559/1015, 55%; difference -13%, 95% CI -17% to -8.3%; p < 0.01). Among men with elevated risk, more GG ≥2 cancers were detected in the MRI group than in the US group (52% vs 43%; difference 9.2%, 95% CI 3.0-15%; p < 0.01). CONCLUSIONS: Risk-adapted US-directed and MRI-directed pathways detected GG ≥2 cancers equally well. The risk-adapted US-directed pathway performs well for prostate cancer diagnosis if prostate MRI capacity and expertise are not available. If prostate MRI availability is sufficient, risk assessment should preferably be performed using MRI, as this avoids more biopsies and detects fewer cases of GG 1 cancer. PATIENT SUMMARY: Among men with suspected prostate cancer, relevant cancers were equally well detected by risk-based pathways using either ultrasound or magnetic resonance imaging (MRI) to guide biopsy of the prostate. If prostate MRI availability is sufficient, risk assessment should be performed with MRI to reduce unnecessary biopsies and detect fewer irrelevant cancers.

Reproducibility of CT-Based Hepatocellular Carcinoma Radiomic Features across Different Contrast Imaging Phases: A Proof of Concept on SORAMIC Trial Data.

Handcrafted radiomic features (HRFs) are quantitative imaging features extracted from regions of interest on medical images which can be correlated with clinical outcomes and biologic characteristics. While HRFs have been used to train predictive and prognostic models, their reproducibility has been reported to be affected by variations in scan acquisition and reconstruction parameters, even within the same imaging vendor. In this work, we evaluated the reproducibility of HRFs across the arterial and portal venous phases of contrast-enhanced computed tomography images depicting hepatocellular carcinomas, as well as the potential of ComBat harmonization to correct for this difference. ComBat harmonization is a method based on Bayesian estimates that was developed for gene expression arrays, and has been investigated as a potential method for harmonizing HRFs. Our results show that the majority of HRFs are not reproducible between the arterial and portal venous imaging phases, yet a number of HRFs could be used interchangeably between those phases. Furthermore, ComBat harmonization increased the number of reproducible HRFs across both phases by 1%. Our results guide the pooling of arterial and venous phases from different patients in an effort to increase cohort size, as well as joint analysis of the phases.

Intra-observer agreements in multidisciplinary team assessments of pancreatic cancer patients.

BACKGROUND AND METHODS: Treatment strategies for pancreatic cancer patients are made by a multidisciplinary team (MDT) board. We aimed to assess intra-observer variance at MDT boards. Participating units staged, assessed resectability, and made treatment allocations for the same patients as they did two years earlier. We disseminated clinical information and CT images of pancreatic cancer patients judged by one MDT board to have nonmetastatic pancreatic cancer to the participating units. All units were asked to re-assess the TNM stage, resectability, and treatment allocation for each patient. To assess intra-observer variance, we computed %-agreements for each participating unit, defined as low (<50%), moderate (50%-75%), and high (>75%) agreement. RESULTS: Eighteen patients were re-assessed by six MDT boards. The overall agreement was moderate for TNM-stage (ranging from 50%-70%) and resectability assessment (53%) but low for treatment allocation (46%). Agreement on resectability assessments was low to moderate. Findings were similar but more pronounced for treatment allocation. We observed a shift in treatment strategy towards increasing use of neoadjuvant chemotherapy, particularly in patients with borderline resectable and locally advanced tumors. CONCLUSIONS: We found substantial intra-observer agreement variations across six different MDT boards of 18 pancreatic cancer patients with two years between the first and second assessment.

Visibility Enhancement and Fog Detection: Solutions Presented in Recent Scientific Papers with Potential for Application to Mobile Systems.

In mobile systems, fog, rain, snow, haze, and sun glare are natural phenomena that can be very dangerous for drivers. In addition to the visibility problem, the driver must face also the choice of speed while driving. The main effects of fog are a decrease in contrast and a fade of color. Rain and snow cause also high perturbation for the driver while glare caused by the sun or by other traffic participants can be very dangerous even for a short period. In the field of autonomous vehicles, visibility is of the utmost importance. To solve this problem, different researchers have approached and offered varied solutions and methods. It is useful to focus on what has been presented in the scientific literature over the past ten years relative to these concerns. This synthesis and technological evolution in the field of sensors, in the field of communications, in data processing, can be the basis of new possibilities for approaching the problems. This paper summarizes the methods and systems found and considered relevant, which estimate or even improve visibility in adverse weather conditions. Searching in the scientific literature, in the last few years, for the preoccupations of the researchers for avoiding the problems of the mobile systems caused by the environmental factors, we found that the fog phenomenon is the most dangerous. Our focus is on the fog phenomenon, and here, we present published research about methods based on image processing, optical power measurement, systems of sensors, etc.

Laser and LIDAR in a System for Visibility Distance Estimation in Fog Conditions.

Visibility is a critical factor for transportation, even if we refer to air, water, or ground transportation. The biggest trend in the automotive industry is autonomous driving, the number of autonomous vehicles will increase exponentially, prompting changes in the industry and user segment. Unfortunately, these vehicles still have some drawbacks and one, always in attention and topical, will be treated in this paper-visibility distance issue in bad weather conditions, particularly in fog. The way and the speed with which vehicles will determine objects, obstacles, pedestrians, or traffic signs, especially in bad visibility, will determine how the vehicle will behave. In this paper, a new experimental set up is featured, for analyzing the effect of the fog when the laser and LIDAR (Light Detection And Ranging) radiation are used in visibility distance estimation on public roads. While using our experimental set up, in the laboratory, the information offered by these measurement systems (laser and LIDAR) are evaluated and compared with results offered by human observers in the same fog conditions. The goal is to validate and unitarily apply the results regarding visibility distance, based on information arrives from different systems that are able to estimate this parameter (in foggy weather conditions). Finally, will be notifying the drivers in case of unexpected situations. It is a combination of stationary and of moving systems. The stationary system will be installed on highways or express roads in areas prone to fog, while the moving systems are, or can be, directly installed on the vehicles (autonomous but also non-autonomous).

Non-invasive imaging prediction of tumor hypoxia: A novel developed and externally validated CT and FDG-PET-based radiomic signatures.

BACKGROUND: Tumor hypoxia increases resistance to radiotherapy and systemic therapy. Our aim was to develop and validate a disease-agnostic and disease-specific CT (+FDG-PET) based radiomics hypoxia classification signature. MATERIAL AND METHODS: A total of 808 patients with imaging data were included: N = 100 training/N = 183 external validation cases for a disease-agnostic CT hypoxia classification signature, N = 76 training/N = 39 validation cases for the H&N CT signature and N = 62 training/N = 36 validation cases for the Lung CT signature. The primary gross tumor volumes (GTV) were manually defined by experts on CT. In order to dichotomize between hypoxic/well-oxygenated tumors a threshold of 20% was used for the [18F]-HX4-derived hypoxic fractions (HF). A random forest (RF)-based machine-learning classifier/regressor was trained to classify patients as hypoxia-positive/ negative based on radiomic features. RESULTS: A 11 feature "disease-agnostic CT model" reached AUC's of respectively 0.78 (95% confidence interval [CI], 0.62-0.94), 0.82 (95% CI, 0.67-0.96) and 0.78 (95% CI, 0.67-0.89) in three external validation datasets. A "disease-agnostic FDG-PET model" reached an AUC of 0.73 (0.95% CI, 0.49-0.97) in validation by combining 5 features. The highest "lung-specific CT model" reached an AUC of 0.80 (0.95% CI, 0.65-0.95) in validation with 4 CT features, while the "H&N-specific CT model" reached an AUC of 0.84 (0.95% CI, 0.64-1.00) in validation with 15 CT features. A tumor volume-alone model was unable to significantly classify patients as hypoxia-positive/ negative. A significant survival split (P = 0.037) was found between CT-classified hypoxia strata in an external H&N cohort (n = 517), while 117 significant hypoxia gene-CT signature feature associations were found in an external lung cohort (n = 80). CONCLUSION: The disease-specific radiomics signatures perform better than the disease agnostic ones. By identifying hypoxic patients our signatures have the potential to enrich interventional hypoxia-targeting trials.

Anaplastic Large Cell T Cell Lymphoma in a Patient With Severe Therapy-refractory Crohn's Disease on Long-standing Immunosuppressive Medication During Ustekinumab Treatment: A Case Report and Review of the Literature.

Use of ustekinumab in Crohn's disease was approved in 2016, and consequently data regarding its real-world safety are still limited. We here present a 29-year-old woman with severe therapy-refractory Crohn's disease, who developed an anaplastic large cell T cell lymphoma during treatment with ustekinumab.

A Survey of Radiation Doses in CT Urography Before and After Implementation of Iterative Reconstruction.

OBJECTIVE: The purpose of this study was to survey the radiation dose used in CT urography (CTU) in routine clinical practice, both before and after implementation of a scanning protocol that uses iterative reconstruction (Adaptive Iterative Dose Reduction 3D [AIDR 3D]). MATERIALS AND METHODS: We retrospectively surveyed dose reports from consecutive CTU examinations performed in 2011 with the use of 64- and 320-MDCT scanners that were reconstructed with filtered back projection (FBP) and from CTU examinations performed from May 2012 through November 2013 that were reconstructed with the use of AIDR 3D. Findings from these dose reports were then correlated with such patient characteristics as weight and body mass index (BMI; weight in kilograms divided by the square of height in meters). Only dose reports from single-bolus three-phase CTU examinations were included in the study. The volume CT dose index, dose-length product (DLP), and effective dose were surveyed both per examination and per phase by use of published effective dose DLP conversion factors. Image quality was evaluated subjectively for a subset of patients. RESULTS: The two study cohorts included 82 patients (median patient weight, 75.0 kg; median BMI, 25.3) who underwent CTU with FBP and 85 patients (median patient weight, 78.0 kg; median BMI, 24.5) who underwent CTU with AIDR 3D. The median total DLP and median effective dose were 924 mGy · cm and 13.0 mSv, respectively, in the CTU with the FBP cohort and 433 mGy · cm and 6.1 mSv, respectively, in the CTU with the AIDR 3D cohort. The median DLP in the unenhanced, nephrogenic, and excretory phases was 218, 300, and 441 mGy · cm, respectively, in patients undergoing CTU with FBP and 114, 121, and 190 mGy · cm, respectively, in patients undergoing CTU with AIDR 3D. Image quality was diagnostic in both groups, with relatively fewer artifacts noted on scans obtained using CTU with AIDR 3D. CONCLUSION: Our study presents detailed dose data from three-phase CTU examinations performed both before and after implementation of iterative reconstruction. Implementation of a CTU protocol using iterative reconstruction resulted in a mean effective dose of 6.1 mSv with preservation of clinical diagnostic image quality.

Small molecule inhibitors of WNT/ß-catenin signaling block IL-1ß- and TNFα-induced cartilage degradation.

INTRODUCTION: In this study, we tested the ability of small molecule inhibitors of WNT/ß-catenin signaling to block interleukin 1ß (IL-1ß)- and tumor necrosis factor α (TNFα)-induced cartilage degradation. Proinflammatory cytokines such as IL-1ß and TNFα are potent inducers of cartilage degradation by upregulating matrix metalloproteinase (MMP) expression and activity. Because WNT/ß-catenin signaling was found to be involved in IL-1ß- and TNFα-induced upregulation of MMP activity, we hypothesized that inhibition of WNT/ß-catenin signaling might block IL-1ß- and TNFα-induced cartilage degradation. We tested the effect of small molecules that block the interaction between ß-catenin and TCF/Lef transcription factors on IL-1ß- and TNFα-induced cartilage degradation in mouse fetal metatarsals. METHODS: We used mouse fetal metatarsals treated with IL-1ß and TNFα as an ex vivo model for cytokine-induced cartilage degradation. Metatarsals were treated with IL-1ß and TNFα in combination with the small molecules PKF115-584, PKF118-310 and CGP049090 at different concentrations and then harvested them for histological and gene expression analysis. RESULTS: We found that IL-1ß- and TNFα-induced cartilage degradation in mouse fetal metatarsals was blocked by inhibiting WNT/ß-catenin signaling using small molecule PKF115-584 and partially using CGP049090 dose-dependently. In addition, we found that PKF115-584 blocked IL-1ß- and TNFα-induced MMP mRNA expression, but did not reverse the inhibitory effect of IL-1ß on the expression of cartilage anabolic genes. CONCLUSION: In this study, we show that inhibition of WNT/ß-catenin signaling by small molecules can effectively prevent IL-1ß- and TNFα-induced cartilage degradation by blocking MMP expression and activity. Furthermore, we elucidate the involvement of WNT/ß-catenin signaling in IL-1ß- and TNFα-induced cartilage degradation.

WNT signaling and cartilage: of mice and men.

In adult articular cartilage, the extracellular matrix is maintained by a balance between the degradation and the synthesis of matrix components. Chondrocytes that sparsely reside in the matrix and rarely proliferate are the key cellular mediators for cartilage homeostasis. There are indications for the involvement of the WNT signaling pathway in maintaining articular cartilage. Various WNTs are involved in the subsequent stages of chondrocyte differentiation during development, and deregulation of WNT signaling was observed in cartilage degeneration. Even though gene expression and protein synthesis can be activated upon injury, articular cartilage has a limited ability of self-repair and efforts to regenerate articular cartilage have so far not been successful. Because WNT signaling was found to be involved in the development and maintenance of cartilage as well as in the degeneration of cartilage, interfering with this pathway might contribute to improving cartilage regeneration. However, most of the studies on elucidating the role of WNT signaling in these processes were conducted using in vitro or in vivo animal models. Discrepancies have been found in the role of WNT signaling between chondrocytes of mouse and human origin, and extrapolation of results from mouse models to the human situation remains a challenge. Elucidation of detailed WNT signaling functions will provide knowledge to improve cartilage regeneration.

Apc bridges Wnt/ß-catenin and BMP signaling during osteoblast differentiation of KS483 cells.

The canonical Wnt signaling pathway influences the differentiation of mesenchymal cell lineages in a quantitative and qualitative fashion depending on the dose of ß-catenin signaling. Adenomatous polyposis coli (Apc) is the critical intracellular regulator of ß-catenin turnover. To better understand the molecular mechanisms underlying the role of Apc in regulating the differentiation capacity of skeletal progenitor cells, we have knocked down Apc in the murine mesenchymal stem cell-like KS483 cells by stable expression of Apc-specific small interfering RNA. In routine culture, KSFrt-Apc(si) cells displayed a mesenchymal-like spindle shape morphology, exhibited markedly decreased proliferation and increased apoptosis. Apc knockdown resulted in upregulation of the Wnt/ß-catenin and the BMP/Smad signaling pathways, but osteogenic differentiation was completely inhibited. This effect could be rescued by adding high concentrations of BMP-7 to the differentiation medium. Furthermore, KSFrt-Apc(si) cells showed no potential to differentiate into chondrocytes or adipocytes. These results demonstrate that Apc is essential for the proliferation, survival and differentiation of KS483 cells. Apc knockdown blocks the osteogenic differentiation of skeletal progenitor cells, a process that can be overruled by high BMP signaling.

Immunogenicity and pharmacokinetic studies of recombinant factor VIII containing lipid cochleates.

Replacement therapy using recombinant factor VIII (rFVIII) is currently the most common therapy for hemophilia A, a bleeding disorder caused by the deficiency of FVIII. However, 15-30% of patients develop inhibitory antibodies against administered rFVIII, which complicates the therapy. Encapsulation or association of protein with lipidic structures can reduce this immune response. Previous studies developed and characterized rFVIII-containing phosphatidylserine (PS) cochleate cylinders using biophysical techniques. It was hypothesized that these structures may provide a reduction in immunogenicity while avoiding the rapid clearance by the reticuloendothelial system (RES) previously observed with liposomal vesicles of similar composition. This study investigated in vivo behavior of the cochleates containing rFVIII including immunogenicity and pharmacokinetics in hemophilia A mice. The rFVIII-cochleate complex significantly reduced the level of inhibitory antibody developed against rFVIII following intravenous (i.v.) administration. Pharmacokinetic modeling allowed assessment of in vivo release kinetics. Cochleates acted as a delayed release delivery vehicle with an input peak of cochleates showed limited RES uptake and associated rFVIII displayed a similar disposition to the free protein upon release from the structure. Incomplete disassociation from the complex limits systemic availability of the protein. Further formulation efforts are warranted to regulate the rate and extent of release of rFVIII from cochleate complexes.

APC mutations are associated with increased bone mineral density in patients with familial adenomatous polyposis.

The canonical Wnt pathway plays a key regulatory role in osteoblastogenesis and bone mass acquisition through its main effector, ß-catenin. Adenomatous polyposis coli (APC) represents the key intracellular gatekeeper of ß-catenin turnover, and heterozygous germ-line mutations in the APC gene cause familial adenomatous polyposis (FAP). Whether APC mutations affect bone mass has not been previously investigated. We conducted a cross-sectional study evaluating skeletal status in FAP patients with a documented APC mutation. Twenty-two FAP patients with a mean age of 42 years (54.5% women) were included in this study. Mean bone mineral density (BMD) Z-scores were significantly increased above normal at all measured sites: lumbar spine (p < .01), total hip (p < .01), femoral neck (p < .05), and trochanter (p < .01). Z-scores were +1 or greater in 14 patients (63.6%) and +2 or greater in 5 (22.7%). Mean values of bone turnover markers were within normal ranges. There was a significant positive correlation between procollagen type I N-terminal propeptide (P1NP) and ß-crosslaps (ß-CTX) (r = 0.70, p < .001) and between these markers and sclerostin and BMD measurements. We demonstrate that FAP patients display a significantly higher than normal mean BMD compared with age- and sex-matched healthy controls in the presence of a balanced bone turnover. Our data suggest a state of "controlled" activation of the Wnt signaling pathway in heterozygous carriers of APC mutations, most likely owing to upregulation of cytoplasmic ß-catenin levels.

Effect of route of administration of human recombinant factor VIII on its immunogenicity in Hemophilia A mice.

Factor VIII is a multi-domain glycoprotein and is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes Hemophilia A, a bleeding disorder. Replacement using exogenous recombinant Factor VIII (FVIII) is the first line of therapy for Hemophilia A. Immunogenicity, the development of binding (total) and neutralizing (inhibitory) antibody against administered protein is a clinical complication of the therapy. There are several product related factors such as presence of aggregates, route and frequency of administration and glycosylation have been shown to contribute to immunogenicity. The effect of route of administration of FVIII on antibody development in Hemophilia A is not completely understood. Here we investigated the effect of route of administration (s.c. or i.v.) on immunogenicity in Hemophilia A mice. The total and inhibitory titers were determined using ELISA and modified Bethesda Assay respectively. The results indicated that s.c. is more immunogenic compared to i.v. route in terms of total antibody titer development (binding antibodies) but no significant differences in inhibitory titer levels could be established.

Role of glycosylation in conformational stability, activity, macromolecular interaction and immunogenicity of recombinant human factor VIII.

Factor VIII (FVIII) is a multi-domain glycoprotein that is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes hemophilia A, a bleeding disorder. Replacement using exogenous recombinant human factor VIII (rFVIII) is the first line of therapy for hemophilia A. The role of glycosylation on the activity, stability, protein-lipid interaction, and immunogenicity of FVIII is not known. In order to investigate the role of glycosylation, a deglycosylated form of FVIII was generated by enzymatic cleavage of carbohydrate chains. The biochemical properties of fully glycosylated and completely deglycosylated forms of rFVIII (degly rFVIII) were compared using enzyme-linked immunosorbent assay, size exclusion chromatography, and clotting activity studies. The biological activity of degly FVIII decreased in comparison to the fully glycosylated protein. The ability of degly rFVIII to interact with phosphatidylserine containing membranes was partly impaired. Data suggested that glycosylation significantly influences the stability and the biologically relevant macromolecular interactions of FVIII. The effect of glycosylation on immunogenicity was investigated in a murine model of hemophilia A. Studies showed that deletion of glycosylation did not increase immunogenicity.

Adenomatous polyposis coli-mediated control of beta-catenin is essential for both chondrogenic and osteogenic differentiation of skeletal precursors.

BACKGROUND: During skeletogenesis, protein levels of beta-catenin in the canonical Wnt signaling pathway determine lineage commitment of skeletal precursor cells to osteoblasts and chondrocytes. Adenomatous polyposis coli (Apc) is a key controller of beta-catenin turnover by down-regulating intracellular levels of beta-catenin. RESULTS: To investigate whether Apc is involved in lineage commitment of skeletal precursor cells, we generated conditional knockout mice lacking functional Apc in Col2a1-expressing cells. In contrast to other models in which an oncogenic variant of beta-catenin was used, our approach resulted in the accumulation of wild type beta-catenin protein due to functional loss of Apc. Conditional homozygous Apc mutant mice died perinatally showing greatly impaired skeletogenesis. All endochondral bones were misshaped and lacked structural integrity. Lack of functional Apc resulted in a pleiotropic skeletal cell phenotype. The majority of the precursor cells lacking Apc failed to differentiate into chondrocytes or osteoblasts. However, skeletal precursor cells in the proximal ribs were able to escape the noxious effect of functional loss of Apc resulting in formation of highly active osteoblasts. Inactivation of Apc in chondrocytes was associated with dedifferentiation of these cells. CONCLUSION: Our data indicate that a tight Apc-mediated control of beta-catenin levels is essential for differentiation of skeletal precursors as well as for the maintenance of a chondrocytic phenotype in a spatio-temporal regulated manner.