Signaling Pathways of AXL Receptor Tyrosine Kinase Contribute to the Pathogenetic Mechanisms of Glioblastoma.

Brain tumors are a diverse collection of neoplasms affecting the brain with a high prevalence rate in people of all ages around the globe. In this pathological context, glioblastoma, a form of glioma that belongs to the IV-grade astrocytoma group, is the most common and most aggressive form of the primary brain tumors. Indeed, despite the best treatments available including surgery, radiotherapy or a pharmacological approach with Temozolomide, glioblastoma patients' mortality is still high, within a few months of diagnosis. Therefore, to increase the chances of these patients surviving, it is critical to keep finding novel treatment opportunities. In the past, efforts to treat glioblastoma have mostly concentrated on customized treatment plans that target specific mutations such as epidermal growth factor receptor (EGFR) mutations, Neurotrophic Tyrosine Receptor Kinase (NTRK) fusions, or multiple receptors using multi-kinase inhibitors like Sunitinib and Regorafenib, with varying degrees of success. Here, we focused on the receptor tyrosine kinase AXL that has been identified as a mediator for tumor progression and therapy resistance in various cancer types, including squamous cell tumors, small cell lung cancer, and breast cancer. Activated AXL leads to a significant increase in tumor proliferation, tumor cell migration, and angiogenesis in different in vitro and in vivo models of cancer since this receptor regulates interplay with apoptotic, angiogenic and inflammatory pathways. Based on these premises, in this review we mainly focused on the role of AXL in the course of glioblastoma, considering its primary biological mechanisms and as a possible target for the application of the most recent treatments.

Recapitulating thyroid cancer histotypes through engineering embryonic stem cells.

Thyroid carcinoma (TC) is the most common malignancy of endocrine organs. The cell subpopulation in the lineage hierarchy that serves as cell of origin for the different TC histotypes is unknown. Human embryonic stem cells (hESCs) with appropriate in vitro stimulation undergo sequential differentiation into thyroid progenitor cells (TPCs-day 22), which maturate into thyrocytes (day 30). Here, we create follicular cell-derived TCs of all the different histotypes based on specific genomic alterations delivered by CRISPR-Cas9 in hESC-derived TPCs. Specifically, TPCs harboring BRAFV600E or NRASQ61R mutations generate papillary or follicular TC, respectively, whereas addition of TP53R248Q generate undifferentiated TCs. Of note, TCs arise by engineering TPCs, whereas mature thyrocytes have a very limited tumorigenic capacity. The same mutations result in teratocarcinomas when delivered in early differentiating hESCs. Tissue Inhibitor of Metalloproteinase 1 (TIMP1)/Matrix metallopeptidase 9 (MMP9)/Cluster of differentiation 44 (CD44) ternary complex, in cooperation with Kisspeptin receptor (KISS1R), is involved in TC initiation and progression. Increasing radioiodine uptake, KISS1R and TIMP1 targeting may represent a therapeutic adjuvant option for undifferentiated TCs.

Therapeutic Potential of Dimethyl Fumarate in Counteract Oral Squamous Cell Carcinoma Progression by Modulating Apoptosis, Oxidative Stress and Epithelial-Mesenchymal Transition.

Oral squamous cell carcinoma (OSCC) is a common human tumor, that originates from buccal mucosa and the tongue, associated with a high mortality rate. Currently, the treatment for OSCC involves surgery, chemotherapy and radiotherapy; however, survival outcomes for OSCC patients remain poor. For this reason, it is necessary to investigate new therapeutic strategies to counteract the progression of OSCC. In this study, we aimed to evaluate the role of dimethyl fumarate (DMF) in modulation of OSCC progression, both in vitro and in an in vivo orthotopic xenograft model. In vitro results revealed that DMF was able to reduce the expression of anti-apoptotic factors as BCL-2 and increased the expression of pro-apoptotic factors as Bax, Caspase-3 and BID. DMF appears to be involved in the modulation of oxidative stress mediators, such as MnSOD and HO-1. Furthermore, DMF showed to reduce the migratory ability of tumor cells and to modulate the expression of markers of epithelial-mesenchymal transition (EMT), as N-cadherin and E-cadherin. The in vivo study confirmed the data obtained in vitro significantly decreasing tumor mass and also reducing oxidative stress and apoptosis. Therefore, based on these results, the use of DMF could be considered a promising strategy to counteract oral cancer progression.

Protein Kinase Inhibitors as a New Target for Immune System Modulation and Brain Cancer Management.

High-grade brain tumors are malignant tumors with poor survival and remain the most difficult tumors to treat. An important contributing factor to the development and progression of brain tumors is their ability to evade the immune system. Several immunotherapeutic strategies including vaccines and checkpoint inhibitors have been studied to improve the effectiveness of the immune system in destroying cancer cells. Recent studies have shown that kinase inhibitors, capable of inhibiting signal transduction cascades that affect cell proliferation, migration, and angiogenesis, have additional immunological effects. In this review, we explain the beneficial therapeutic effects of novel small-molecule kinase inhibitors and explore how, through different mechanisms, they increase the protective antitumor immune response in high-grade brain tumors.

Overview on Common Genes Involved in the Onset of Glioma and on the Role of Migraine as Risk Factor: Predictive Biomarkers or Therapeutic Targets?

Gliomas are relatively rare but fatal cancers, and there has been insufficient research to specifically evaluate the role of headache as a risk factor. Nowadays, gliomas are difficult to cure due to the infiltrative nature and the absence of specific adjuvant therapies. Until now, mutations in hundreds of genes have been identified in gliomas and most relevant discoveries showed specific genes alterations related to migraine as potential risk factors for brain tumor onset. Prognostic biomarkers are required at the time of diagnosis to better adapt therapies for cancer patients. In this review, we aimed to highlight the significant modulation of CLOCK, BMLA1 and NOTCH genes in glioma onset and development, praising these genes to be good as potentially attractive therapeutic markers for brain tumors. A improved knowledge regarding the role of these genes in triggering or modulating glioma maybe the key to early diagnosing brain tumor onset in patients affected by a simple headache. In addition, investigating on these genes we can suggest potential therapeutic targets for treating brain tumors. These considerations open up the possibility of personalized treatments that can target each brain tumor's specific genetic abnormality.

Destroying the Shield of Cancer Stem Cells: Natural Compounds as Promising Players in Cancer Therapy.

In a scenario where eco-sustainability and a reduction in chemotherapeutic drug waste are certainly a prerogative to safeguard the biosphere, the use of natural products (NPs) represents an alternative therapeutic approach to counteract cancer diseases. The presence of a heterogeneous cancer stem cell (CSC) population within a tumor bulk is related to disease recurrence and therapy resistance. For this reason, CSC targeting presents a promising strategy for hampering cancer recurrence. Increasing evidence shows that NPs can inhibit crucial signaling pathways involved in the maintenance of CSC stemness and sensitize CSCs to standard chemotherapeutic treatments. Moreover, their limited toxicity and low costs for large-scale production could accelerate the use of NPs in clinical settings. In this review, we will summarize the most relevant studies regarding the effects of NPs derived from major natural sources, e.g., food, botanical, and marine species, on CSCs, elucidating their use in pre-clinical and clinical studies.

GSK343, an Inhibitor of Enhancer of Zeste Homolog 2, Reduces Glioblastoma Progression through Inflammatory Process Modulation: Focus on Canonical and Non-Canonical NF-κB/IκBα Pathways.

Glioblastoma (GB) is a tumor of the central nervous system characterized by high proliferation and invasiveness. The standard treatment for GB includes radiotherapy and chemotherapy; however, new therapies are needed. Particular attention was given to the role of histone methyltransferase enhancer of zeste-homolog-2 (EZH2) in GB. Recently, several EZH2-inhibitors have been developed, particularly GSK343 is well-known to regulate apoptosis and autophagy processes; however, its abilities to modulate canonical/non-canonical NF-κB/IκBα pathways or an immune response in GB have not yet been investigated. Therefore, this study investigated for the first time the effect of GSK343 on canonical/non-canonical NF-κB/IκBα pathways and the immune response, by an in vitro, in vivo and ex vivo model of GB. In vitro results demonstrated that GSK343 treatments 1, 10 and 25 µM significantly reduced GB cell viability, showing the modulation of canonical/non-canonical NF-κB/IκBα pathway activation. In vivo GSK343 reduced subcutaneous tumor mass, regulating canonical/non-canonical NF-κB/IκBα pathway activation and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD). Ex vivo results confirmed the anti-proliferative effect of GSK343 and also demonstrated its ability to regulate immune response through CXCL9, CXCL10 and CXCL11 expression in GB. Thus, GSK343 could represent a therapeutic strategy to counteract GB progression, thanks to its ability to modulate canonical/non-canonical NF-κB/IκBα pathways and immune response.

A Case of Non-Irradiated Balloon Cell Melanoma of the Choroid: Expanding the Morphological Spectrum of Primary Uveal Melanomas.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults and usually has a very poor prognosis. Histologically, UMs have been classified in epithelioid cell type, spindle cell type, and mixed cell type. Balloon cells are large pale cells that contain small, hyperchromatic, central nuclei with vesiculated, clear, and lipid-rich cytoplasm. A balloon cell morphology is infrequently observed in naevi and even less frequently in malignant melanomas of the skin, conjunctiva, ciliary body and choroid. In this regard, UMs that exhibit balloon cell features are generally those previously treated with proton beam irradiation and then enucleated, rather than those that directly underwent primary surgery. To the best of our knowledge, very few cases of primary UM showing extensive balloon cell morphology have been reported in scientific literature to date. We herein present an unusual case of primary UM with diffuse balloon cell changes in a 69-year-old woman.

Glioblastoma, IDH-Wild Type With FGFR3-TACC3 Fusion: When Morphology May Reliably Predict the Molecular Profile of a Tumor. A Case Report and Literature Review.

It has been reported that in-frame FGFR3-TACC3 fusions confer to glioblastomas, IDH-wild type (GBMs, IDHwt) some unusual morphologic features, including monomorphous rounded cells with ovoid nuclei, nuclear palisading, endocrinoid network of "chicken-wire" vessels, microcalcifications and desmoplastic stroma, whose observation may predict the molecular profile of the tumor. We herein present a case of recurrent GBMs, IDHwt, exhibiting some of the above-mentioned morphological features and a molecularly-proven FGFR3-TACC3 fusion. A 56-year-old man presented to our hospital for a recurrent GBM, IDHwt, surgically treated at another center. Histologically, the tumor, in addition to the conventional GBM morphology, exhibited the following peculiar morphologic features: (1) monomorphous neoplastic cells with rounded nuclei and scant pale cytoplasm; (2) thin capillary-like vessels with "chicken-wire" pattern; (3) nuclear palisading; (4) formation of vague perivascular pseudorosettes; (5) spindled tumor cells embedded in a loose, myxoid background. Based on this unusual morphology, molecular analyses were performed and an FGFR3 exon17-TACC3 exon 10 fusion was found. The present case contributes to widening the morphologic spectrum of FGFR3-TACC3-fused GBM, IDHwt and emphasizes that pathologists, in the presence of a GBM, IDHwt with unconventional morphology, should promptly search for this fusion gene.

Potential Therapeutic Effects of PPAR Ligands in Glioblastoma.

Glioblastoma (GB), also known as grade IV astrocytoma, represents the most aggressive form of brain tumor, characterized by extraordinary heterogeneity and high invasiveness and mortality. Thus, a great deal of interest is currently being directed to investigate a new therapeutic strategy and in recent years, the research has focused its attention on the evaluation of the anticancer effects of some drugs already in use for other diseases. This is the case of peroxisome proliferator-activated receptors (PPARs) ligands, which over the years have been revealed to possess anticancer properties. PPARs belong to the nuclear receptor superfamily and are divided into three main subtypes: PPAR-α, PPAR-ß/δ, and PPAR-γ. These receptors, once activated by specific natural or synthetic ligands, translocate to the nucleus and dimerize with the retinoid X receptors (RXR), starting the signal transduction of numerous genes involved in many physiological processes. PPARs receptors are activated by specific ligands and participate principally in the preservation of homeostasis and in lipid and glucose metabolism. In fact, synthetic PPAR-α agonists, such as fibrates, are drugs currently in use for the clinical treatment of hypertriglyceridemia, while PPAR-γ agonists, including thiazolidinediones (TZDs), are known as insulin-sensitizing drugs. In this review, we will analyze the role of PPARs receptors in the progression of tumorigenesis and the action of PPARs agonists in promoting, or not, the induction of cell death in GB cells, highlighting the conflicting opinions present in the literature.

The Biological Function of MicroRNAs in Bone Tumors.

Micro ribonucleic acids (miRNAs) are small endogenous noncoding RNAs molecules that regulate gene expression post-transcriptionally. A single miRNA is able to target hundreds of specific messenger RNA (mRNAs) by binding to the 3'-untranslated regions. miRNAs regulate different biological processes such as cell proliferation, differentiation and apoptosis. Altered miRNA expression is certainly related to the development of the most common human diseases, including tumors. Osteosarcoma (OS), Ewing's Sarcoma (ES), and Chondrosarcoma (CS) are the most common primary bone tumors which affect mainly children and adolescents. A significant dysregulation of miRNA expression, in particular of mir-34, mir-21, mir-106, mir-143, and miR-100, has been revealed in OS, ES and CS. In this context, miRNAs can act as either tumor suppressor genes or oncogenes, contributing to the initiation and progression of bone tumors. The in-depth study of these small molecules can thus help to better understand their biological functions in bone tumors. Therefore, this review aims to examine the potential role of miRNAs in bone tumors, especially OS, ES and CS, and to suggest their possible use as potential therapeutic targets for the treatment of bone tumors and as biomarkers for early diagnosis.

Beneficial effect of KYP-2047, a propyl-oligopeptidase inhibitor, on oral squamous cell carcinoma.

Oral squamous cell-carcinoma (OSCC) is a common cancer which arises from the alveolar ridge, buccal mucosa, and tongue. Among OSCC, the incidence of tongue squamous cell-carcinoma (TSCC) is growing all over the world. Oral carcinogenesis has been linked to genetic mutations, chromosomal aberrations and viral factors. Apoptosis and angiogenesis play a key role in the development of oral cancer. Therefore, it is very important discover new therapeutic strategies to counteract oral cancer progression. This study aimed to investigate the effect of KYP-2047 in an in vitro model of TSCC and in vivo CAL27-xenograft model. Our results demonstrated that KYP-2047 was able to reduce TSCCs cell viability at the concentrations of 50 µM and 100 µM. Additionally, KYP-2047 was able to increase Bax, Bad and caspase-3 expression, whereas Bcl-2 and p53 expression were reduced. Moreover, KYP-2047 significantly reduced vascular-endothelial-growth-factor (VEGF) and endothelial-nitric-oxide-synthase (eNOS) expression. In the vivo xenograft model, KYP-2047 at doses of 1 and 5 mg/kg significantly reduced tumor burden and tumor weight, decreasing also angiogenesis markers VEGF and eNOS. Moreover, KYP-2047 increased Bax and reduced Bcl2 expressions. Thus, KYP-2047 could represent a potential therapeutic treatment to counteract tongue oral-cancer growth, thanks its abilities to modulate angiogenesis and apoptosis pathways.

Targeting Phosphatases and Kinases: How to Checkmate Cancer.

Metastatic disease represents the major cause of death in oncologic patients worldwide. Accumulating evidence have highlighted the relevance of a small population of cancer cells, named cancer stem cells (CSCs), in the resistance to therapies, as well as cancer recurrence and metastasis. Standard anti-cancer treatments are not always conclusively curative, posing an urgent need to discover new targets for an effective therapy. Kinases and phosphatases are implicated in many cellular processes, such as proliferation, differentiation and oncogenic transformation. These proteins are crucial regulators of intracellular signaling pathways mediating multiple cellular activities. Therefore, alterations in kinases and phosphatases functionality is a hallmark of cancer. Notwithstanding the role of kinases and phosphatases in cancer has been widely investigated, their aberrant activation in the compartment of CSCs is nowadays being explored as new potential Achille's heel to strike. Here, we provide a comprehensive overview of the major protein kinases and phosphatases pathways by which CSCs can evade normal physiological constraints on survival, growth, and invasion. Moreover, we discuss the potential of inhibitors of these proteins in counteracting CSCs expansion during cancer development and progression.

NLRP3 Inflammasome Inhibitor BAY-117082 Reduces Oral Squamous Cell Carcinoma Progression.

Oral cancer is one of the most common human malignancies, and its incidence is increasing worldwide. In particular, oral squamous cell carcinoma (OSCC) is characterized by high rates of proliferation, invasiveness, and metastasis. Currently, standard treatment for OSCC includes surgical removal, chemotherapy, and radiotherapy; however, the survival rate of patients with OSCC remains low, thus new therapies are needed. It has been proven that excessive NLRP3 inflammasome activation and apoptosis alteration may contribute to oral cancer progression. This study aimed to investigate the effect of BAY-117082, an NLRP3 inflammasome inhibitor, in an in vitro and in vivo xenograft model of oral cancer. In vitro results revealed that BAY-117082 at concentrations of 5, 10, and 30 µM was able to reduce OSCC cell viability. BAY-117082 at higher concentrations significantly reduced NLRP3, ASC, caspase-1, IL-1ß, and IL-18 expression. Moreover, Bax, Bad, and p53 expression were increased, whereas Bcl-2 expression was reduced. Furthermore, the in vivo study demonstrated that BAY-117082 at doses of 2.5 and 5 mg/kg significantly decreased subcutaneous tumor mass, and also reduced NLRP3 inflammasome pathway activation. Therefore, based on these results, the use of BAY-117082 could be considered a promising strategy to counteract oral cancer progression, thanks its ability to modulate the NLRP3 inflammasome and apoptosis pathways.

Clear Cell Carcinoma Arising in an Abdominal Wall Cesarean Section Scar: A Case Report With Description of Pathological and Molecular Features.

Clear cell carcinoma is a clinically and biologically distinct type of carcinoma predominantly encountered in the ovary and endometrium. In the ovary, it is frequently associated with endometriosis, which is a well-known risk factor. Endometriosis has often been described in the abdominal wall of women who had a cesarean section; however, malignant transformation is a very rare event, occurring in <1% of cases. The authors report a case involving a 52-year-old woman with an abdominal wall nodule at a cesarean section scar. Radiology revealed a mass, measuring 8 cm in size, in the abdominal wall suggestive of a soft tissue tumor. After resection, histology revealed the presence of clear, eosinophilic, and hobnail cells, which, together with immunohistochemical and molecular findings, enabled the diagnosis of clear cell carcinoma of the abdominal wall. The present report describes the clinical, radiological, pathological, and molecular features of an unusual abdominal lesion that represents a rare but challenging diagnosis.

Medullary Carcinoma of the Gastrointestinal Tract: Report on Two Cases with Immunohistochemical and Molecular Features.

Medullary carcinoma of the colon is a rare histological variant characterized by a poorly differentiated morphology, an aberrant immunophenotype, and microsatellite instability. Despite the lack of glandular differentiation, medullary carcinoma is reported to have a good prognosis. It is typically located in the right colon and frequently affects older women. Due to its clinical, histological, biological, and genetic peculiarity, medullary carcinoma requires an accurate diagnosis and the awareness of this diagnostic possibility. We describe the morphological, immunohistochemical, and molecular findings of two interesting cases, the first one in the right colon of a patient and the second one in the terminal ileum of a patient with Crohn's disease. Deeper knowledge of all the biological and clinical features will allow appropriate and specific treatment of this tumor in the future.

Cancer Stem Cell Biomarkers Predictive of Radiotherapy Response in Rectal Cancer: A Systematic Review.

BACKGROUND: Rectal cancer (RC) is one of the most commonly diagnosed and particularly challenging tumours to treat due to its location in the pelvis and close proximity to critical genitourinary organs. Radiotherapy (RT) is recognised as a key component of therapeutic strategy to treat RC, promoting the downsizing and downstaging of large RCs in neoadjuvant settings, although its therapeutic effect is limited due to radioresistance. Evidence from experimental and clinical studies indicates that the likelihood of achieving local tumour control by RT depends on the complete eradication of cancer stem cells (CSC), a minority subset of tumour cells with stemness properties. METHODS: A systematic literature review was conducted by querying two scientific databases (Pubmed and Scopus). The search was restricted to papers published from 2009 to 2021. RESULTS: After assessing the quality and the risk of bias, a total of 11 studies were selected as they mainly focused on biomarkers predictive of RT-response in CSCs isolated from patients affected by RC. Specifically these studies showed that elevated levels of CD133, CD44, ALDH1, Lgr5 and G9a are associated with RT-resistance and poor prognosis. CONCLUSIONS: This review aimed to provide an overview of the current scenario of in vitro and in vivo studies evaluating the biomarkers predictive of RT-response in CSCs derived from RC patients.

Adipose stem cell niche reprograms the colorectal cancer stem cell metastatic machinery.

Obesity is a strong risk factor for cancer progression, posing obesity-related cancer as one of the leading causes of death. Nevertheless, the molecular mechanisms that endow cancer cells with metastatic properties in patients affected by obesity remain unexplored.Here, we show that IL-6 and HGF, secreted by tumor neighboring visceral adipose stromal cells (V-ASCs), expand the metastatic colorectal (CR) cancer cell compartment (CD44v6 + ), which in turn secretes neurotrophins such as NGF and NT-3, and recruits adipose stem cells within tumor mass. Visceral adipose-derived factors promote vasculogenesis and the onset of metastatic dissemination by activation of STAT3, which inhibits miR-200a and enhances ZEB2 expression, effectively reprogramming CRC cells into a highly metastatic phenotype. Notably, obesity-associated tumor microenvironment provokes a transition in the transcriptomic expression profile of cells derived from the epithelial consensus molecular subtype (CMS2) CRC patients towards a mesenchymal subtype (CMS4). STAT3 pathway inhibition reduces ZEB2 expression and abrogates the metastatic growth sustained by adipose-released proteins. Together, our data suggest that targeting adipose factors in colorectal cancer patients with obesity may represent a therapeutic strategy for preventing metastatic disease.

Messing Up the Cancer Stem Cell Chemoresistance Mechanisms Supported by Tumor Microenvironment.

Despite the recent advances in cancer patient management and in the development of targeted therapies, systemic chemotherapy is currently used as a first-line treatment for many cancer types. After an initial partial response, patients become refractory to standard therapy fostering rapid tumor progression. Compelling evidence highlights that the resistance to chemotherapeutic regimens is a peculiarity of a subpopulation of cancer cells within tumor mass, known as cancer stem cells (CSCs). This cellular compartment is endowed with tumor-initiating and metastasis formation capabilities. CSC chemoresistance is sustained by a plethora of grow factors and cytokines released by neighboring tumor microenvironment (TME), which is mainly composed by adipocytes, cancer-associated fibroblasts (CAFs), immune and endothelial cells. TME strengthens CSC refractoriness to standard and targeted therapies by enhancing survival signaling pathways, DNA repair machinery, expression of drug efflux transporters and anti-apoptotic proteins. In the last years many efforts have been made to understand CSC-TME crosstalk and develop therapeutic strategy halting this interplay. Here, we report the combinatorial approaches, which perturb the interaction network between CSCs and the different component of TME.

VECTOR: An Integrated Correlation Network Database for the Identification of CeRNA Axes in Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults and, although its genetic background has been extensively studied, little is known about the contribution of non-coding RNAs (ncRNAs) to its pathogenesis. Indeed, its competitive endogenous RNA (ceRNA) regulatory network comprising microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and mRNAs has been insufficiently explored. Thanks to UM findings from The Cancer Genome Atlas (TCGA), it is now possible to statistically elaborate these data to identify the expression relationships among RNAs and correlative interaction data. In the present work, we propose the VECTOR (uVeal mElanoma Correlation NeTwORk) database, an interactive tool that identifies and visualizes the relationships among RNA molecules, based on the ceRNA model. The VECTOR database contains: i) the TCGA-derived expression correlation values of miRNA-mRNA, miRNA-lncRNA and lncRNA-mRNA pairs combined with predicted or validated RNA-RNA interactions; ii) data of sense-antisense sequence overlapping; iii) correlation values of Transcription Factor (TF)-miRNA, TF-lncRNA, and TF-mRNA pairs associated with ChiPseq data; iv) expression data of miRNAs, lncRNAs and mRNAs both in UM and physiological tissues. The VECTOR web interface can be queried, by inputting the gene name, to retrieve all the information about RNA signaling and visualize this as a graph. Finally, VECTOR provides a very detailed picture of ceRNA networks in UM and could be a very useful tool for researchers studying RNA signaling in UM. The web version of Vector is freely available at the URL reported at the end of the Introduction.