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Accurate brain tumor segmentation on magnetic resonance imaging (MRI) has wide-ranging applications such as radiosurgery planning. Advances in artificial intelligence, especially deep learning (DL), allow development of automatic segmentation that overcome the labor-intensive and operator-dependent manual segmentation. We aimed to evaluate the accuracy of the top-performing DL model from the 2018 Brain Tumor Segmentation (BraTS) challenge, the impact of missing MRI sequences, and whether a model trained on gliomas can accurately segment other brain tumor types. We trained the model using Medical Decathlon dataset, applied it to the BraTS 2019 glioma dataset, and developed additional models using individual and multimodal MRI sequences. The Dice score was calculated to assess the model’s accuracy compared to ground truth labels by neuroradiologists on BraTS dataset. The model was then applied to a local dataset of 105 brain tumors, performance of which was qualitatively evaluated. The DL model using pre- and post-gadolinium contrast T1 and T2 FLAIR sequences performed best, with a Dice score 0.878 for whole tumor, 0.732 tumor core, and 0.699 active tumor. Lack of T1 or T2 sequences did not significantly degrade performance, but FLAIR and T1C were important contributors. All segmentations performed by the model in the local dataset, including non-glioma cases, were considered accurate by a pool of specialists. The DL model could use available MRI sequences to optimize glioma segmentation and adopt transfer learning to segment non-glioma tumors, thereby serving as a useful tool to improve treatment planning and personalized surveillance of patients.

Mutations in isocitrate dehydrogenase genes IDH1 and IDH2 are frequently found in diffuse and anaplastic astrocytic and oligodendroglial tumours as well as in secondary glioblastomas. As IDH is a very important prognostic, diagnostic and therapeutic biomarker for glioma, it is of paramount importance to determine its mutational status. The haematoxylin and eosin (H&E) staining is a valuable tool in precision oncology as it guides histopathology-based diagnosis and proceeding patient’s treatment. However, H&E staining alone does not determine the IDH mutational status of a tumour. Deep learning methods applied to MRI data have been demonstrated to be a useful tool in IDH status prediction, however the effectiveness of deep learning on H&E slides in the clinical setting has not been investigated so far. Furthermore, the performance of deep learning methods in medical imaging has been practically limited by small sample sizes currently available. Here we propose a data augmentation method based on the Generative Adversarial Networks (GAN) deep learning methodology, to improve the prediction performance of IDH mutational status using H&E slides. The H&E slides were acquired from 266 grade II-IV glioma patients from a mixture of public and private databases, including 130 IDH-wildtype and 136 IDH-mutant patients. A baseline deep learning model without data augmentation achieved an accuracy of 0.794 (AUC = 0.920). With GAN-based data augmentation, the accuracy of the IDH mutational status prediction was improved to 0.853 (AUC = 0.927) when the 3,000 GAN generated training samples were added to the original training set (24,000 samples). By integrating also patients’ age into the model, the accuracy improved further to 0.882 (AUC = 0.931). Our findings show that deep learning methodology, enhanced by GAN data augmentation, can support physicians in gliomas’ IDH status prediction.

Susceptibility-weighted imaging (SWI) is a technique useful for evaluation of the internal structures of brain tumors, including microvasculature and microbleeds. Intratumoral patterns of magnetic susceptibility can be quantified by means of fractal analysis. Here, we propose a radiomics methodological pipeline to merge advanced fractal-based computational modelling with statistical analysis to objectively characterize the fingerprint of gliomas and brain metastases. Forty-seven patients with glioma (grades II-IV, according to the WHO 2016 classification system) and fourteen with brain metastases underwent 3 Tesla MRI using a SWI protocol. All images underwent computational analysis aimed to quantify three Euclidean parameters (related to tumor and SWI volume) and five fractal-based parameters (related to the pixel distribution and geometrical complexity of the SWI patterns). Principal components analysis, linear and quadratic discriminant analysis, K-nearest neighbor and support vector machine methods were used to discriminate between tumor types. The combination of parameters offered an objective evaluation of the SWI pattern in gliomas and brain metastases. The model accurately predicted 88% of glioblastoma, according to the quantification of intratumoral SWI features, failing to discriminate the other types. SWI is not normally used to classify brain tumors, however fractal-based multi-parametric computational analysis can be used to characterize intratumoral SWI patterns to objectively quantify tumors-related features. Specific parameters still have to be identified to provide completely automatic computerized differential diagnosis.

To determine the heterogeneity of glucose uptake applying fractal analysis on positron emission tomography/computed tomography (PET/CT) with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) images in patients with non-small cell lung carcinoma (NSCLC) before surgery, and to assess whether this heterogeneity was associated with disease-free survival (DFS). [18F]FDG PET/CT scans of 113 patients’ prior surgery were retrospectively revised. PET DICOM images were analyzed for fractal geometry using a ad hoc software to automatically determine the following indexes: (a) mean intensity value (MIV), (b) standard deviation (SD), (c) relative dispersion (RD), (d) three-dimensional (3D) histogram of the fractal dimension (3D HIST FR DIM), and (e) fractal dimension in 3D (3D-FD). All the fractal indexes were subsequently compared with metabolic parameters and disease-free survival (DFS). We found a significant correlation between 3D-FD and SUVmax, SUVmean, MTV, and TLG. Additionally, positive correlations between MIV, SD, and all metabolic parameters were also detected. Patients with high 3D-FD tumor (≥ 1.62) showed significantly higher values of SUVmax, SUVmean, MTV, and TLG than those with lower 3D-FD. In univariate analysis, median 3D-FD and median TLG were significantly associated with DFS (p = 0.04 and p = 0.03, respectively). These findings were confirmed on log-rank test. On multivariate analysis, among age, stage disease, histotype, 3D-FD, and metabolic parameters, only 3D-FD was identified as independent prognostic factor for DFS (p = 0.032; HR 0.418, 95 % CI 0.189–0.926). 3D-FD was different between adenocarcinoma and squamous cell carcinoma (1.60 versus 1.88, p = 0.014), and 3D-FD value was found higher in advanced stage disease. Metabolic heterogeneity determined applying fractal principles on PET images can be considered as a novel imaging biomarker for survival in patients with NSCLC.

This study aimed to determine the prognostic role of textural features and their association with metabolic parameters, hypoxia, and cancer-related immune markers in non-small-cell lung cancer (NSCLC) patients.The trial was registered at http://www.clinicaltrials.gov (NCT02519062). From January 2010 to May 2014, 44 patients (male : female=33 : 11; median age: 69.5 years), referred to our Institution for NSCLC resection, were enrolled. Tumor specimens were assessed for HIF-1α, CD68-TAMs, CD8-TILs, PD-1-TILs, and PD-L1 expressions. All patients underwent fluorine-18-fluorodeoxyglucose (F-FDG) PET before surgery. Semiquantitative parameters included maximum standardized uptake value (SUVmax), SUVpeak, SUVmean, metabolic tumor volume, and total lesion glycolysis, whereas for heterogeneity, we considered tumor sphericity, skewness, kurtosis, entropy, and energy. Parameters were correlated with disease-free survival (DFS) considering a median follow-up of 22.7 months.SUVmax (cutoff: 7.9; P=0.015), SUVpeak (cutoff: 6.7; P=0.013), SUVmean (cutoff: 5.5; P=0.028), metabolic tumor volume (cutoff: 3.6 cm; P=0.027), and entropy (cutoff: 1.89; P=0.045) showed a statistically significant association with DFS. Also, a high expression of cytoplasmic HIF-1α (score 3) was associated with DFS (hazard ratio: 0.09; P=0.003). All F-FDG PET variables differed significantly in tumors with high or low entropy (≤1.89). Also, a significantly higher level of mean CD8-TILs was observed in tumors with higher entropy (P=0.041).Using identified prognostic factors, we developed a scoring system, which was confirmed to be associated with DFS (P0.004). On receiver operating characteristics analysis, a score above 3 was defined as the optimal cutoff point.Tumor heterogeneity, metabolic parameters, and high expression of hypoxia were found to be prognostic factors in NSCLC patients who were candidates for surgery. Higher levels of entropy appear to be associated with increased density of CD8-TILs. The combination of investigated prognostic factors enabled the development of a potential scoring system associated with DFS.

Background: We developed a macrophage-based treatment relying on ex vivo transduction of autologous hematopoietic stem and progenitor cells (HSPC) to express immune-payloads within the TME. Our ATMP (Temferon) targets IFN-a, an immune-modulatory molecule counteracting also neo-angiogenesis and tumor growth, to a subset of Tie2-expressing, tumor-infiltrating macrophages known as TEMs. Materials and Methods: TEM-GBM is an open-label, Phase I/IIa dose-escalation study evaluating safety and efficacy of Temferon in up to 21 newly diagnosed glioblastoma patients with unmethylated MGMT promoter. Key eligibility criteria include age 18-70 years, ECOG 0-1 and KPS >70%, and adequate cardiac, renal, hepatic and pulmonary function. Important exclusion criteria include the presence of active autoimmune disease or receipt of any oral or parenteral chemotherapy or immunotherapy within 2 years of screening. Autologous CD34+ HSPC are mobilized with lenograstim and plerixafor, collected by apheresis, purified and transduced ex vivo with a 3 rd generation lentiviral vector encoding for IFN-a2. Transgene expression is confined to TEMs by the Tie2 promoter and post-transcriptional regulation by microRNA-126 thus achieving tumor specificity. The study evaluates safety and biological activity of Temferon in 7 cohorts of three patients each, where escalating doses of Temferon are co-administered with a fixed CD34+ cell dose of non-manipulated supporter cells following a sub-myeloablative conditioning regimen (Thiotepa + BCNU or + Busulfan). The primary endpoints for this study are: Engraftment of Temferon over the first 90 DaysThe proportion of patients achieving hematologic recovery by Day +30 from ASCTShort-term tolerability of Temferon; stable blood counts and absence of cytopenias, absence of significant organ toxicities (> grade 2); absence of Replication Competent Lentivirus The figure below reports the TEM-GBM study design. Results: As of 28th June 2021, 18 patients have been enrolled; 15 received Temferon (D+0) with follow-up of 30 - 697 days. There was rapid engraftment and hematological recovery after the conditioning regimen. Median neutrophil and platelet engraftment occurred at D+13 and D+12 for patients in cohort 1-3 and D+16 and D+15 for patients assigned to cohort 4 and 5, respectively. Temferon-derived differentiated cells, as determined by the presence of vector genomes in the DNA of peripheral blood and bone marrow cells, were found within 14 days post treatment and persisted subsequently, albeit at lower levels (up to 18 months). Very low concentrations of IFNa were detected in the plasma (average 7.8 pg/ml at D+30; baseline < LLOQ) and in the cerebrospinal fluid (average 1.6 pg/ml at D+30; baseline < LLOQ), suggesting tight regulation of transgene expression. Seven deaths occurred: six at D+241, +322, +340, +402, +478, +646 after Temferon administration due to disease progression, and one at D+60 due to complications following the conditioning regimen. Nine patients had progressive disease (PD; range D-12 to +239). SAEs include infections, venous thromboembolism, brain abscess, hemiparesis, GGT elevation and poor performance status compatible with autologous stem cell transplantation, concomitant medications and PD. Four patients underwent second surgery. These recurrent tumors had gene-marked cells present and increased expression of IFN-responsive gene signatures compared to diagnosis, indicative of local IFNa release by TEMs. In one patient, a stable lesion (as defined by MRI) had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased IFN-response signature than in a progressing lesion. The T-cell immune repertoire changed with evidence for expansion of tumor-associated clones. Tumor microenvironment characterization by scRNA and TCR sequencing is ongoing. Conclusion: These interim results show that Temferon is generally well tolerated by patients, with no dose limiting toxicities identified to date. The results provide initial evidence of Temferon's potential to activate the immune system and reprogram the tumor microenvironment (TME), as predicted by preclinical studies. Figure 1 Figure 1. Disclosures Naldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Computational Neurosurgery is a novel field where computational modeling and artificial intelligence (AI) are used to analyze diseases of neurosurgical interest. Our aim is to apply AI models to brain tumor (BT) images to a) automatically segment BTs on pre-operative MRI, b) predict the genetic subtype of glioma on intra- and post-operative histological specimens; and c) predict the extent of resection according to connectomics data. For the segmentation task, we used 510 BT images to train a deep learning (DL) model for automatic segmentation of the tumors’ edges and comparison of the AI-generated masks versus experts’ consensus (quantified by means of the dice score). For the histopathology task, we digitalized 266 hematoxylin/eosin slides of gliomas (including 130 IDH-wildtype and 136 IDH-mutant) and applied a DL architecture to predict the IDH genetic status, then validated by immunohistochemistry and genetic sequencing. The datasets were also augmented by generating synthetic glioma images by means of a Generative Adversarial Network methodology. The resection of 10 BTs was also customized according to connectomics data. In the segmentation experiment, we reached a dice score of ~0.9 (out of 1.0), while further demonstrating that only the T1, T1 after gadolinium, and FLAIR sequences are necessary for accurate automatic segmentation. In the histopathology task, we were able to predict the genetic status with accuracy between 76% and 95% using the DL model. The machine learning-based connectome analysis allowed us to perform safe supramaximal resection. We have shown the robustness of applying AI methodology or the automatic segmentation of BTs in MR imaging. Moreover, we have also shown that AI can be used to predict the genetic status, specifically, IDH, in histopathology images of gliomas. Our results support the use of AI in the clinical scenario for a fast and objective computerized characterization of patients affected by BTs.

Temferon is an ex vivo gene therapy consisting of autologous HSPCs genetically modified to deliver IFN-α2 within the tumor microenvironment (TME) by Tie-2 expressing macrophages. TEM-GBM is an open-label, Phase I/IIa dose-escalation study evaluating safety and efficacy of Temferon in up to 21 newly diagnosed GBM patients with unmethylated MGMT. Autologous HSPCs are transduced with a LVV encoding for IFN-a2 gene. As of 30th April 2021, 18 patients have been enrolled; 13 received Temferon (D+0) with follow-up of 8 – 662 days. After conditioning and Temferon infusion, a rapid engraftment and hematological recovery occurred, with median neutrophil and platelet engraftment at D+13 and D+12, respectively. No dose limiting toxicities were reported. Temferon-derived cells were found within 14 days post treatment and persisted albeit at lower levels in the long-term. Five deaths occurred: one at +478, three at +322, +340 and +402 days due to PD, and the fourth at +60 due to complications following the conditioning regimen. Eight patients had PD (-12 to +239). SAEs include respiratory tract infections, pulmonary embolism, CMV and C.Diff infections, febrile neutropenia, hemiparesis, seizure, brain abscess, worsening of performance status and respiratory failure compatible with ASCT, concomitant medications and PD. Four patients underwent second surgery. Recurrent tumors had gene-marked cells present and increased expression of ISGs compared to diagnosis, indicative of local IFNa release by TEMs. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased IFN-response signature than in a progressing lesion. Characterization of T-cell immune repertoire suggests the expansion of tumor-associated clones. TME characterization by scRNA and TCR sequencing is ongoing. Interim results show that Temferon is well tolerated, with no dose limiting toxicities identified to date and provide initial evidence of potential immune system activation within the TME.

Temferon is a macrophage-based treatment relying on ex-vivo transduction of autologous HSPCs to express immune-payloads within the TME. Temferon targets the immune-modulatory molecule IFN-a, to a subset of tumor infiltrating macrophages known as Tie-2 expressing macrophages (TEMs) due to the Tie2 promoter and a post-transcriptional regulation layer represented by miRNA-126 target sequences. As of 31st May 2021, 15-patients received Temferon (D+0) with follow-up of 3 – 693 days. After conditioning neutrophil and platelet engraftment occurred at D+13 and D+13.5, respectively. Temferon-derived differentiated cells, as determined be the number of vector copy per genome, were found within 14 days post treatment and persisted albeit at lower levels up to 18-months. Very low concentrations of IFN-a in the plasma (8.7 pg/ml-D+30) and in the CSF (1.6 pg/ml-D+30) were detected, suggesting tight regulation of transgene expression. Five-deaths occurred at D+322, +340, +402, +478 and +646 due to PD, and one at D+60 due to complications following the conditioning regimen. Eight-patients had progressive disease (range: D-11 to +239) as expected for this tumor type. SAEs include GGT elevation (possibly related to Temferon) and infections, venous thromboembolism, brain abscess, hemiparesis, seizures, anemia and general physical condition deterioration, compatible with ASCT, concomitant medications and PD. Four-patients underwent 2ndsurgery. Recurrent tumors had gene-marked cells and increased expression of ISGs compared to first surgery, indicative of local IFNa release by TEMs. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased ISGs than in the progressing lesion, detected in the same patient. Tumor-associated clones expanded in the periphery. TME characterization by scRNA and TCR-sequencing is ongoing. To date, Temferon is well tolerated, with no DLTs identified. The results provide initial evidence of Temferon potential to activate the immune system of GBM patients, as predicted by preclinical studies.

OBJECTIVE: The aim of this study was to analyse early and delayed acquisition on copper-64 diacetyl-bisN4-methylthiosemicarbazone (Cu-ATSM) PET/CT in a small cohort of patients by comparing semiquantitative and computer-aided fractal geometry analyses. PATIENTS AND METHODS: Five cancer patients, including non-small-cell lung cancer and head and neck cancer, were investigated with Cu-ATSM PET/CT. Participants received an intravenous injection of Cu-ATSM according to body size and were imaged 60 min (early) and 16 h (delayed) later on hybrid PET/CT. Reconstructed images were visualized on advanced workstations for the definition of semiquantitative parameters: standardized uptake value (SUV)max, SUVratio-to-muscle, SUVmean, hypoxic volume (HV) and hypoxic burden (HB=HV×SUVmean). DICOM data retrieved from both scans were analysed using an ad-hoc computer program to determine the mean intensity value, SD, relative dispersion, three-dimensional histogram fractal dimension and three-dimensional fractal dimension. RESULTS: All tumour lesions showed increased uptake of Cu-ATSM at early evaluation, with a median SUVratio-to-muscle of 4.42 (range: 1.58-5.62), a median SUVmax of 5.3 (range: 1.9-7.3), a median SUVmean of 2.8 (range: 1.5-3.9), a median HV of 41.6 cm (range: 2.8-453.7) and a median HB of 161.5 cm (range: 4.4-1112.5). All semiquantitative data obtained at 1 h were consistent with the parameters obtained on delayed imaging (P>0.05). A borderline statistically significant difference was found only for SUVmax of the muscle (P=0.045). Fractal geometry analysis on DICOM images showed that all parameters at early imaging showed no statistically significant difference with late acquisition (P>0.05). CONCLUSION: Our findings support the consistency of Cu-ATSM PET/CT images obtained at early and delayed acquisition for the assessment of tumour lesions.

2532 Background: Genetically modified cell-based therapies are relevant in immuno-oncology due to their potential for tumor specificity & potential durability. We developed a cell-based treatment, Temferon, relying on ex-vivo transduction of autologous HSPCs to express therapeutic payloads within the tumor microenvironment. Temferon targets IFNa to Tie-2 expressing macrophages (TEMs). Methods: TEM-GBM is an open-label, Phase I/IIa dose-escalation study evaluating safety & efficacy of Temferon in up to 21 newly diagnosed patients with glioblastoma & unmethylated MGMT promoter. Autologous HSPCs are transduced ex-vivo with a lentiviral vector encoding for IFNa. The transgene expression is confined to TEMs due to the Tie2 promoter & the post-transcriptional regulation by miRNA-126. Results: As of January 17 2021, 15 patients have been enrolled; 9 received Temferon (D+0) with follow-up of 61 – 559 days. There was rapid engraftment & hematological recovery after the conditioning regimen. Median neutrophil & platelet engraftment occurred at D+13 & D+12, respectively. Temferon-derived differentiated cells, as determined by the presence of vector genomes in the DNA of peripheral blood & bone marrow cells, were found within 14 days post treatment & persisted subsequently, albeit at lower levels (up to 18 months). We also detected very low concentrations of IFNa in the plasma (median 5pg/ml at D+30; baseline < LLOQ) & in the cerebrospinal fluid, suggesting tight regulation of transgene expression. Three deaths occurred: two at D+343 & +402 after Temferon administration due to disease progression, & one at D+60 due to complications following the conditioning regimen. Seven patients had progressive disease (PD; range D+27-239) as expected for this tumor type. SAEs include infections, venous thromboembolism, brain abscess, hemiparesis, GGT elevation & poor performance status compatible with autologous stem cell transplantation, concomitant medications & PD. Four patients underwent second surgery. These recurrent tumors had gene-marked cells present & increased expression of IFN-responsive gene signatures compared to diagnosis, indicative of local IFNa release by TEMs. In one patient a stable lesion (as defined by MRI) had a higher proportion of T cells & TEMs within the myeloid infiltrate & an increased IFN-response signature than in a progressing lesion. The T-cell immune repertoire changed with evidence for expansion of tumor-associated clones. Tumor microenvironment characterization by scRNA & TCR sequencing is ongoing. Conclusions: Our interim results show that Temferon is well tolerated by patients, with no dose limiting toxicities identified to date. The results provide initial evidence of Temferon potential to modulate the TME of GBM patients, as predicted by preclinical studies. Clinical trial information: NCT03866109.

We find that student performance on questions posed in the standard heterogeneous combination of algebraic direct demand and graphic inverse demand is significantly worse than their performance on questions posed in homogeneous combinations. Since this performance deficit persists with advanced students, it seems that economists' canonical presentation of demand may hinder, rather than help, learning. We recommend that Principles students begin with the homogenous, direct combination of algebra and graph before turning to the standard direct-inverse combination. This modification would create benefits on the extensive margin — reducing attrition from confusion — and intensive margin — increasing comprehension for all students.

Hepatitis C virus (HCV) is still one of the major causes of chronic viral infection worldwide, and hepatic steatosis is a frequent pathological finding in patients with chronic HCV-related diseases. It is unclear whether the steatosis is associated with host factors or the virus itself, although a consistent relationship has been found between steatosis and a necro-inflammatory reaction with the increased secretion of immuno-regulators. A primary sources of inflammatory mediators are mast cells (MCs) bone marrow-derived cells that are detected in both normal and diseased livers. We determined MC density and correlated it with the fibrosis, inflammatory reaction and steatosis observed in the liver biopsies of patients affected by HCV with or without steatosis. All the histological features were assessed using a computer-aided image analysis system. There was a statistically significant difference in MC density between the HCV-infected patients with and without steatosis, with the lower mean value being detected in those without (P < 0.02). Furthermore, a nonstatistically significant difference in fibrosis and inflammation between the two patient groups was found. In conclusion, this is the first study showing a significant increase in MC density in the tissues of patients with chronic HCV infection and histologically documented steatosis.

Hepatocellular carcinoma (HCC) remains one of the major public health problems throughout the world. Although originally associated with tumorigenic processes, liver angiogenesis has also been observed in the context of different liver inflammatory, fibrotic, and ischemic conditions. Here we investigate the fractal dimension as a quantitator of non-Euclidean two-dimensional vascular geometry in a series of paired specimens of primary HCC and surrounding non-tumoral tissue, and discuss why this parameter might provide additional information regarding cancer behavior. The application of fractal geometry to the measurement of liver vascularity and the availability of a computer-aided quantitative method can eliminate errors in visual interpretation, and make it possible to obtain closer-to-reality numerals that are compulsory for any measurement process.

Mast cells (MCs) are multifunctional effector cells of the immune system. MCs were originally thought to be involved in IgE-associated immediate hypersensitivity and allergic disorders, but it is now known that they contain or elaborate an array of mediators with a multitude of effects on many other cells. A number of studies have found that MCs are involved in various liver diseases. Although still controversial, they seem to be involved in the liver's fibrotic response to chronic inflammation and parasitic infection. Hepatic fibrosis is the most frequent liver response to toxic, infectious, or metabolic agents. During the establishment of this pathological condition, there is an increase in the components of the basement membrane that leads to continuous basement membrane-like structures being raised within Disse's space and a decrease in the number of sinusoid endothelial fenestrae. This leads to a complex process called "sinusoidal capillarization." At the cellular level, liver fibrogenesis is initiated by hepatocyte necrosis, which induces the recruitment of a large number of inflammatory cells, including MCs, which can be considered the primary effectors of the process changing sinusoidal endothelial cells into capillary-type endothelial cells. We review the roles played by MCs in hepatic chronic diseases and describe a biopsy section of hepatic tissue taken from a patient with chronic C virus-related hepatitis showing diffuse sinusoidal capillarization and a high density of MCs. This observation has led us to hypothesize a relationship between these highly specialized cells and sinusoidal capillarization.

Hereditary angioedema (HAE) is a potentially life-threatening autosomal dominant deficiency affecting 1 in 50,000 individuals from all ethnic groups worldwide. More than 99% of HAE cases are caused by a deficiency in functional plasma C1 esterase inhibitor (C1E-INH, a serine protease inhibitor) due to mutations in the SERPING1 gene. Low plasma C1E-INH activity dysregulates the contact, complement, and fibrinolytic systems, resulting in unpredictable, recurrent submucosal edema of cutaneous tissues, gastrointestinal and respiratory tracts. If not treated in a timely manner, laryngeal edema can result in death by asphyxiation. Current HAE treatments consist of management of acute attacks, repeated prophylactic therapy with C1E-INH or long term prophylaxis with attenuated androgens, each complicated by a high economic burden, limited compliance, drug side effects and contraindications. To circumvent this challenge, we hypothesized that a one-time administration of an adeno-associated virus (AAV) gene transfer vector expressing the genetic sequence of C1 esterase-inhibitor (serotype 10 expressing the human CIE-INH coding sequence, AAVrh.10hC1EI) would provide sustained C1E-INH activity levels in plasma, sufficient to prevent angioedema episodes. To study the efficacy of AAVrh.10hC1EI, using CRISPR/Cas9 technology we created a novel C1E-INH deficient mouse model analogous to human HAE disease, and characterized the resulting SERPING1 gene mutations by genome sequencing. The heterozygous mouse model shares characteristics associated with HAE in humans including decreased C1E-INH and C4 levels in plasma and increased vascular permeability. Administration of AAVrh.10hC1EI to the heterozygous mice resulted in sustained human C1E levels above the predicted therapeutic levels. In order to demonstrate that the increased vascular permeability observed in heterozygote C1E-INH+/- mice was a direct result of C1E-INH deficiency, Evans blue dye was injected intravenously and extravasation of dye from the vasculature evaluated. Compared to wild type mice under baseline conditions, the C1EINH+/- mice had increased extravasation of the dye into the hind paws quantitated by optical absorbance at 600 nm. Strikingly, AAVrh10.hC1EI-treated (1011 gc) mice displayed a marked decrease in dye extravasation, whereas non-treated C1E-INH +/- mice had markedly increased dye extravasation. These results demonstrate that a single treatment with AAVrh.10hC1EI has the potential to provide long term protection from angioedema attacks in the affected population, representing a paradigm shift in current therapeutic approaches.

It is well known that angiogenesis is a complex process that accompanies neoplastic growth, but pituitary tumours are less vascularized than normal pituitary glands. Several analytical methods aimed at quantifying the vascular system in two-dimensional histological sections have been proposed, with very discordant results. In this study we investigated the non-Euclidean geometrical complexity of the two-dimensional microvasculature of normal pituitary glands and pituitary adenomas by quantifying the surface fractal dimension that measures its space-filling property. We found a statistical significant difference between the mean vascular surface fractal dimension estimated in normal versus adenomatous tissues (P = 0.01), normal versus secreting adenomatous tissues (P = 0.0003), and normal versus non-secreting adenomatous tissues (P = 0.047), whereas the difference between the secreting and non-secreting adenomatous tissues was not statistically significant. This study provides the first demonstration that fractal dimension is an objective and valid quantitator of the two-dimensional geometrical complexity of the pituitary gland microvascular network in physiological and pathological states. Further studies are needed to compare the vascular surface fractal dimension estimates in different subtypes of pituitary tumours and correlate them with clinical parameters in order to evaluate whether the distribution pattern of vascular growth is related to a particular state of the pituitary gland.

Background Cancer remains one of the most complex diseases affecting humans and, despite the impressive advances that have been made in molecular and cell biology, how cancer cells progress through carcinogenesis and acquire their metastatic ability is still widely debated. Conclusion There is no doubt that human carcinogenesis is a dynamic process that depends on a large number of variables and is regulated at multiple spatial and temporal scales. Viewing cancer as a system that is dynamically complex in time and space will, however, probably reveal more about its underlying behavioural characteristics. It is encouraging that mathematicians, biologists and clinicians continue to contribute together towards a common quantitative understanding of cancer complexity. This way of thinking may further help to clarify concepts, interpret new and old experimental data, indicate alternative experiments and categorize the acquired knowledge on the basis of the similarities and/or shared behaviours of very different tumours.

AIM: To provide the accurate alternative metrical means of monitoring the effects of new antiviral drugs on the reversal of newly formed collagen. METHODS: Digitized histological biopsy sections taken from 209 patients with chronic C virus hepatitis with different grade of fibrosis or cirrhosis, were measured by means of a new, rapid, user-friendly, fully computer-aided method based on the international system meter rectified using fractal principles. RESULTS: The following were described: geometric perimeter, area and wrinkledness of fibrosis; the collation of the Knodell, Sheuer, Ishak and METAVIR scores with fractal-rectified metric measurements; the meaning of the physical composition of fibrosis in relation to the magnitude of collagen islets; the intra- and inter-biopsy sample variability of these parameters; the “staging” of biopsy sections indicating the pathway covered by fibrosis formation towards its maximum known value; the quantitative liver tissue architectural changes with the Hurst exponent. CONCLUSION: Our model provides the first metrical evaluations of the geometric properties of fibrosis and the quantitative architectural changes of the liver tissue. The representativeness of histological sections of the whole liver is also discussed in the light of the results obtained with the Hurst coefficient.

Primary biliary cirrhosis, is an autoimmune disease, semiquantitatively described by four stages obtained from evaluating the stage of hepatic structures. The follovng pathologic events, inflammation, destruction-regeneration of biliary tissue CK7+ and fibrosis determine this disease.

AIM: To describe a quantitative analysis method for liver biopsy sections with a machine that we have named “Dioguardi Histological Metriser” which automatically measures the residual hepatocyte mass (including hepatocytes vacuolization), inflammation, fibrosis and the loss of liver tissue tectonics. METHODS: We analysed digitized images of liver biopsy sections taken from 398 patients. The analysis with Dioguardi Histological Metriser was validated by comparison with semi-quantitative scoring system. RESULTS: The method provides: (1) the metrical extension in two-dimensions (the plane) of the residual hepatocellular set, including the area of vacuoles pertinent to abnormal lipid accumulation; (2) the geometric measure of the inflammation basin, which distinguishes intra-basin space and extra-basin dispersed parenchymal leukocytes; (3) the magnitude of collagen islets, (which were considered truncated fractals and classified into three degrees of magnitude); and (4) the tectonic index that quantifies alterations (disorders) in the organization of liver tissue. Dioguardi Histological Metriser machine allows to work at a speed of 0.1 mm2/s, scanning a whole section in 6-8 min. CONCLUSION: The results are the first standardized metrical evaluation of the geometric properties of the parenchyma, inflammation, fibrosis, and alterations in liver tissue tectonics of the biopsy sections. The present study confirms that biopsies are still valuable, not only for diagnosing chronic hepatitis, but also for quantifying changes in the organization and order of liver tissue structure.

AIM: To assess the sampling variability of computer-aided, fractal-corrected measures of fibrosis in liver biopsies. METHODS: Samples were derived from six to eight different parts of livers removed from 12 patients with clinically and histologically proven cirrhosis undergoing orthotopic liver transplantation. Sirius red-stained sections with a thickness of 2 μm were digitized using a computer-aided image analysis system that automatically measures the surface of fibrosis, as well as its outline perimeter, fractal surface and outline dimensions, wrinkledness, and Hurst coefficient. RESULTS: We found a high degree of inter-sample variability in the measurements of the surface [coefficient of variation (CV) = 43% ± 13%] and wrinkledness (CV = 28% ± 9%) of fibrosis, but the inter-sample variability of Hurst’s exponent was low (CV = 14% ± 2%). CONCLUSION: This study suggests that Hurst’s exponent might be used in clinical practice as the best histological estimate of fibrosis in the whole organ, and evidences the fact that biopsy sections, which are fundamental for the qualitative diagnosis of chronic hepatitis, play a key role in the quantitative estimate of architectural changes in liver tissue.

Streptococcal mitogen (SM), an extracellular product of group A streptococci, is non specifically mitogenic for both B and T lymphocytes. The mitogenic activity of SM is resistant to digestion with trypsin, to heating at 100 °C for 5 min, and to treatment with dithiothreitol. The proliferative response of lymphocytes from patients with a history of rheumatic fever is similar to that of lymphocytes from healthy donors when stimulated with optimal concentrations of SM, but is significantly reduced when low doses of SM are used. T lymphocytes stimulated with SM acquire Ia antigens and the ability to stimulate allogeneic and autologous lymphocytes in mixed lymphocyte reactions. An involvement of Ia antigens in these reactions is indicated by the specific block by monoclonal antibodies to human Ia antigens.

T lymphocytes from patients with solid tumors and from aged donors are abnormal in their expression of Ia antigens following in vitro stimulation with phytohemagglutinin (PHA). Ia antigens were not detected on PHA-activated T lymphocytes from 15 of 27 patients with solid tumors. The abnormality in T lymphocytes from 25 donors older than 60 years was evidenced by a reduction in the percentage of T cells acquiring Ia antigens following stimulation with suboptimal amounts of PHA and by a delayed appearance of those antigens. In both groups of donors the defect in Ia antigen expression by PHA-activated T cells did not correlate with the reduced [3H]thymidine uptake. PHA-activated T cells from aged donors and from patients with solid tumors were poorly stimulatory in autologous and allogeneic mixed lymphocyte reactions. Furthermore, T lymphocytes from these two groups of donors displayed a reduced proliferative response to autologous non-T cells, but a normal proliferative response to allogeneic PHA-activated T cells and to non-T cells from control subjects.

Human T lymphocytes activated with PHA express Ia-like antigens and acquire the ability to stimulate autologous T lymphocytes in mixed lymphocyte reaction. This reaction is immunological in nature since it has specificity and memory. Ia-like antigens play a role in the stimulation of T lymphocytes by autologous PHA-T lymphocytes since monoclonal antibodies to Ia-like antigens can significantly, although not completely, inhibit the stimulation.

Guancydine was administered to 10 patients for a period of 3 weeks to 6 months. The drug produced a decrease in both supine and standing blood pressures as well as in digital vascular reactivity but caused no change in tritiated norepinephrine uptake. Its action appears to be direct inhibition of vascular smooth muscle. Side effects were minimal and transitory.