Severe high-molecular-weight kininogen deficiency: clinical characteristics, deficiency-causing KNG1 variants, and estimated prevalence.

BACKGROUND: Severe high-molecular-weight kininogen (HK) deficiency is a poorly studied autosomal recessive contact system defect caused by pathogenic, biallelic KNG1 variants. AIM: We performed the first comprehensive analysis of diagnostic, clinical, genetic, and epidemiological aspects of HK deficiency. METHODS: We collected clinical information and blood samples from a newly detected HK-deficient individual and from published cases identified by a systematic literature review. Activity and antigen levels of coagulation factors were determined. Genetic analyses of KNG1 and KLKB1 were performed by Sanger sequencing. The frequency of HK deficiency was estimated considering truncating KNG1 variants from GnomAD. RESULTS: We identified 48 cases of severe HK deficiency (41 families), of these 47 have been previously published (n = 19 from gray literature). We genotyped 3 cases and critically appraised 10 studies with genetic data. Ten HK deficiency-causing variants (one new) were identified. All of them were truncating mutations, whereas the only known HK amino acid substitution with a relevant phenotype instead causes hereditary angioedema. Conservative estimates suggest an overall prevalence of severe HK deficiency of approximately one case per 8 million population, slightly higher in Africans. Individuals with HK deficiency appeared asymptomatic and had decreased levels of prekallikrein and factor XI, which could lead to misdiagnosis. CONCLUSION: HK deficiency is a rare condition with only few known pathogenic variants. It has an apparently good prognosis but is prone to misdiagnosis. Our understanding of its clinical implications is still limited, and an international prekallikrein and HK deficiency registry is being established to fill this knowledge gap.

Decellularized esophageal tubular scaffold microperforated by quantum molecular resonance technology and seeded with mesenchymal stromal cells for tissue engineering esophageal regeneration.

Current surgical options for patients requiring esophageal replacement suffer from several limitations and do not assure a satisfactory quality of life. Tissue engineering techniques for the creation of customized "self-developing" esophageal substitutes, which are obtained by seeding autologous cells on artificial or natural scaffolds, allow simplifying surgical procedures and achieving good clinical outcomes. In this context, an appealing approach is based on the exploitation of decellularized tissues as biological matrices to be colonized by the appropriate cell types to regenerate the desired organs. With specific regard to the esophagus, the presence of a thick connective texture in the decellularized scaffold hampers an adequate penetration and spatial distribution of cells. In the present work, the Quantum Molecular Resonance® (QMR) technology was used to create a regular microchannel structure inside the connective tissue of full-thickness decellularized tubular porcine esophagi to facilitate a diffuse and uniform spreading of seeded mesenchymal stromal cells within the scaffold. Esophageal samples were thoroughly characterized before and after decellularization and microperforation in terms of residual DNA content, matrix composition, structure and biomechanical features. The scaffold was seeded with mesenchymal stromal cells under dynamic conditions, to assess the ability to be repopulated before its implantation in a large animal model. At the end of the procedure, they resemble the original esophagus, preserving the characteristic multilayer composition and maintaining biomechanical properties adequate for surgery. After the sacrifice we had histological and immunohistochemical evidence of the full-thickness regeneration of the esophageal wall, resembling the native organ. These results suggest the QMR microperforated decellularized esophageal scaffold as a promising device for esophagus regeneration in patients needing esophageal substitution.

Circulating Extracellular Vesicles Impair Mesenchymal Stromal Cell Differentiation Favoring Adipogenic Rather than Osteogenic Differentiation in Adolescents with Obesity.

Excess body weight has been considered beneficial to bone health because of its anabolic effect on bone formation; however, this results in a poor quality bone structure. In this context, we evaluated the involvement of circulating extracellular vesicles in the impairment of the bone phenotype associated with obesity. Circulating extracellular vesicles were collected from the plasma of participants with normal weight, as well as overweight and obese participants, quantified by flow cytometry analysis and used to treat mesenchymal stromal cells and osteoblasts to assess their effect on cell differentiation and activity. Children with obesity had the highest amount of circulating extracellular vesicles compared to controls. The treatment of mesenchymal stromal cells with extracellular vesicles from obese participants led to an adipogenic differentiation in comparison to vesicles from controls. Mature osteoblasts treated with extracellular vesicles from obese participants showed a reduction in differentiation markers in comparison to controls. Children with obesity who regularly performed physical exercise had a lower circulating extracellular vesicle amount in comparison to those with a sedentary lifestyle. This pilot study demonstrates how the high amount of circulating extracellular vesicles in children with obesity affects the bone phenotype and that physical activity can partially rescue this phenotype.

Identification of neuroblastoma cell lines with uncommon TAZ+/mesenchymal stromal cell phenotype with strong suppressive activity on natural killer cells.

BACKGROUND: Neuroblastoma (NB) is the most common, extracranial childhood solid tumor arising from neural crest progenitor cells and is a primary cause of death in pediatric patients. In solid tumors, stromal elements recruited or generated by the cancer cells favor the development of an immune-suppressive microenvironment. Herein, we investigated in NB cell lines and in NB biopsies, the presence of cancer cells with mesenchymal phenotype and determined the immune-suppressive properties of these tumor cells on natural killer (NK) cells. METHODS: We assessed the mesenchymal stromal cell (MSC)-like phenotype and function of five human NB cell lines and the presence of this particular subset of neuroblasts in NB biopsies using flow-cytometry, immunohistochemistry, RT-qPCR, cytotoxicity assays, western blot and silencing strategy. We corroborated our data consulting a public gene-expression dataset. RESULTS: Two NB cell lines, SK-N-AS and SK-N-BE(2)C, exhibited an unprecedented MSC phenotype (CD105+/CD90+/CD73+/CD29+/CD146+/GD2+/TAZ+). In these NB-MSCs, the ectoenzyme CD73 and the oncogenic/immune-regulatory transcriptional coactivator TAZ were peculiar markers. Their MSC-like nature was confirmed by their adipogenic and osteogenic differentiation potential. Immunohistochemical analysis confirmed the presence of neuroblasts with MSC phenotype (CD105+/CD73+/TAZ+). Moreover, a public gene-expression dataset revealed that, in stage IV NB, a higher expression of TAZ and CD105 strongly correlated with a poorer outcome.Among the NB-cell lines analyzed, only NB-MSCs exhibited multifactorial resistance to NK-mediated lysis, inhibition of activating NK receptors, signal adaptors and of NK-cell cytotoxicity through cell-cell contact mediated mechanisms. The latter property was controlled partially by TAZ, since its silencing in NB cells efficiently rescued NK-cell cytotoxic activity, while its overexpression induced opposite effects in non-NB-MSC cells. CONCLUSIONS: We identified a novel NB immunoregulatory subset that: (i) displayed phenotypic and functional properties of MSC, (ii) mediated multifactorial resistance to NK-cell-induced killing and (iii) efficiently inhibited, in coculture, the cytotoxic activity of NK cells against target cells through a TAZ-dependent mechanism. These findings indicate that targeting novel cellular and molecular components may disrupt the immunomodulatory milieu of the NB microenvironment ameliorating the response to conventional treatments as well as to advanced immunotherapeutic approaches, including adoptive transfer of NK cells and chimeric antigen receptor T or NK cells.

Neuroblastoma-secreted exosomes carrying miR-375 promote osteogenic differentiation of bone-marrow mesenchymal stromal cells.

Bone marrow (BM) is the major target organ for neuroblastoma (NB) metastasis and its involvement is associated with poor outcome. Yet, the mechanism by which NB cells invade BM is largely unknown. Tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (MSCs) have been recognized as a fundamental part of the associated tumour stroma. Here, we show that BM-MSCs isolated from NB patients with BM involvement exhibit a greater osteogenic potential than MSCs from non-infiltrated BM. We show that BM metastasis-derived NB-cell lines secrete higher levels of exosomal miR-375, which promotes osteogenic differentiation in MSCs. Of note, clinical data demonstrate that high level of miR-375 correlates with BM metastasis in NB patients. Our findings suggest, indeed, a potential role for exosomal miR-375 in determining a favourable microenvironment in BM to promote metastatic progression. MiR-375 may, thus, represent a novel biomarker and a potential target for NB patients with BM involvement.

Successful muscle regeneration by a homologous microperforated scaffold seeded with autologous mesenchymal stromal cells in a porcine esophageal substitution model.

BACKGROUND: Since the esophagus has no redundancy, congenital and acquired esophageal diseases often require esophageal substitution, with complicated surgery and intestinal or gastric transposition. Peri-and-post-operative complications are frequent, with major problems related to the food transit and reflux. During the last years tissue engineering products became an interesting therapeutic alternative for esophageal replacement, since they could mimic the organ structure and potentially help to restore the native functions and physiology. The use of acellular matrices pre-seeded with cells showed promising results for esophageal replacement approaches, but cell homing and adhesion to the scaffold remain an important issue and were investigated. METHODS: A porcine esophageal substitute constituted of a decellularized scaffold seeded with autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs) was developed. In order to improve cell seeding and distribution throughout the scaffolds, they were micro-perforated by Quantum Molecular Resonance (QMR) technology (Telea Electronic Engineering). RESULTS: The treatment created a microporous network and cells were able to colonize both outer and inner layers of the scaffolds. Non seeded (NSS) and BM-MSCs seeded scaffolds (SS) were implanted on the thoracic esophagus of 4 and 8 pigs respectively, substituting only the muscle layer in a mucosal sparing technique. After 3 months from surgery, we observed an esophageal substenosis in 2/4 NSS pigs and in 6/8 SS pigs and a non-practicable stricture in 1/4 NSS pigs and 2/8 SS pigs. All the animals exhibited a normal weight increase, except one case in the SS group. Actin and desmin staining of the post-implant scaffolds evidenced the regeneration of a muscular layer from one anastomosis to another in the SS group but not in the NSS one. CONCLUSIONS: A muscle esophageal substitute starting from a porcine scaffold was developed and it was fully repopulated by BM-MSCs after seeding. The substitute was able to recapitulate in shape and function the original esophageal muscle layer.

Severe plasma prekallikrein deficiency: Clinical characteristics, novel KLKB1 mutations, and estimated prevalence.

BACKGROUND: Severe plasma prekallikrein (PK) deficiency is an autosomal-recessive defect characterized by isolated activated partial thromboplastin time prolongation. To date, no comprehensive methodologically firm analysis has investigated the diagnostic, clinical, and genetic characteristics of PK deficiency, and its prevalence remains unknown. PATIENTS/METHODS: We described new families with PK deficiency, retrieved clinical and laboratory information of cases systematically searched in the (gray) literature, and collected blood of these cases for complementary analyses. The Genome Aggregation Database (gnomAD) and the population-based Gutenberg Health Study served to study the prevalence of mutations and relevant genetic variants. RESULTS: We assembled a cohort of 111 cases from 89 families and performed new genetic analyses in eight families (three unpublished). We identified new KLKB1 mutations, excluded the pathogenicity of some of the previously described ones, and estimated a prevalence of severe PK deficiency of 1/155 668 overall and 1/4725 among Africans. One individual reported with PK deficiency had, in fact, congenital kininogen deficiency associated with decreased PK activity. One quarter of individuals had factor XII clotting activity below the reference range. Four major bleeding events were described in 96 individuals, of which 3 were provoked, for a prevalence of 4% and an annualized rate of 0.1%. The prevalence of cardiovascular events was 15% (6% <40 years; 21% 40-65 years; 33% >65 years) for an annualized rate of 0.4%. CONCLUSIONS: We characterized the genetic background of severe PK deficiency, critically appraised mutations, and provided prevalence estimates. Our data on laboratory characteristics and clinical course of severe PK deficiency may have clinical implications.

Coagulation in Brain Tumors: Biological Basis and Clinical Implications.

Cancer patients commonly show abnormal laboratory coagulation tests, indicating a subclinical hypercoagulable condition that contribute to morbidity and mortality. The hypercoagulation status not only increases the risk of thromboembolic events but also influences the tumor biology promoting its growth and progression by stimulating intracellular signaling pathways. Recent molecular studies characterized the role of oncogene and suppressor gene in activating clotting pathways, as an integral feature of the neoplastic transformation. It is now clear how haemostatic processes, activated by cancer cells harboring oncogenic mutations, rely on the molecular profile of a particular malignancy, an aspect particularly evident in the differential coagulome profiles showed by different molecular subtypes of brain tumors, such as glioblastoma and medulloblastoma. This review focuses on the biological and clinical aspects of haemostasis in cancer with particular regard on brain tumors.

Phenotypic and functional characterization of mesenchymal stromal cells isolated from pediatric patients with severe idiopathic nephrotic syndrome.

BACKGROUND: Idiopathic nephrotic syndrome (INS) is one of the most common renal diseases in the pediatric population; considering the role of the immune system in its pathogenesis, corticosteroids are used as first-line immunosuppressive treatment. Due to its chronic nature and tendency to relapse, a significant proportion of children experience co-morbidity due to prolonged exposure to corticosteroids and concomitant immunosuppression with second-line, steroid-sparing agents. Mesenchymal stromal cells (MSCs) are multipotent cells that represent a key component of the bone marrow (BM) microenvironment; given their unique immunoregulatory properties, their clinical use may be exploited as an alternative therapeutic approach in INS treatment. METHODS: In view of the possibility of exploiting their immunoregulatory properties, we performed a phenotypical and functional characterization of MSCs isolated from BM of five INS patients (INS-MSCs; median age, 13 years; range, 11-16 years) in comparison with MSCs isolated from eight healthy donors (HD-MSCs). MSCs were expanded ex vivo and then analyzed for their properties. RESULTS: Morphology, proliferative capacity, immunophenotype and differentiation potential did not differ between INS-MSCs and HD-MSCs. In an allogeneic setting, INS-MSCs were able to prevent both T- and B-cell proliferation and plasma-cell differentiation. In an in-vitro model of experimental damage to podocytes, co-culture with INS-MSCs appeared to be protective. DISCUSSION: Our results demonstrate that INS-MSCs maintain the main biological and functional properties typical of HD-MSCs; these data suggest that MSCs may be used in autologous cellular therapy approaches for INS treatment.

Biological and functional characterization of bone marrow-derived mesenchymal stromal cells from patients affected by primary immunodeficiency.

Mesenchymal stromal cells (MSCs) represent a key component of bone marrow (BM) microenvironment and display immune-regulatory properties. We performed a detailed analysis of biological/functional properties of BM-MSCs derived from 33 pediatric patients affected by primary immune-deficiencies (PID-MSCs): 7 Chronic Granulomatous Disease (CGD), 15 Wiskott-Aldrich Syndrome (WAS), 11 Severe Combined Immunodeficiency (SCID). Results were compared with MSCs from 15 age-matched pediatric healthy-donors (HD-MSCs). Clonogenic and proliferative capacity, differentiation ability, immunophenotype, immunomodulatory properties were analyzed. WB and RT-qPCR for CYBB, WAS and ADA genes were performed. All PID-MSCs displayed clonogenic and proliferative capacity, morphology and immunophenotype comparable with HD-MSCs. PID-MSCs maintained the inhibitory effect on T- and B-lymphocyte proliferation, except for decreased inhibitory ability of SCID-MSCs at MSC:PBMC ratio 1:10. While HD- and CGD-MSCs were able to inhibit monocyte maturation into immature dendritic cells, in SCID- and WAS-MSCs this ability was reduced. After Toll-like Receptor priming, PID-MSCs displayed in vitro an altered gene expression profile of pro- and anti-inflammatory soluble factors. PID-MSCs displayed lower PPARγ levels and WAS- and SCID-MSCs higher levels of key osteogenic markers, as compared with HD-MSCs. Our results indicate that PID-MSCs may be defective in some functional abilities; whether these defects contribute to disease pathophysiology deserves further investigation.

Proteomic Analysis of Neuroblastoma-Derived Exosomes: New Insights into a Metastatic Signature.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Around 70% of patients with metastatic disease at diagnosis present bone-marrow infiltration, which is considered a marker of poor outcome; however, the mechanism underlying this specific tropism has to be elucidated. Tumor-derived exosomes may support metastatic progression in several tumors by interacting with the microenvironment, and may serve as tumor biomarkers. The main objective of this study is to identify an exosomal signature associated with NB metastatic bone-marrow dissemination. Therefore, the proteomic cargo of exosomes isolated from NB cell lines derived from primary tumor and bone-marrow metastasis is characterized. The comparison among exosomal proteins show 15 proteins exclusively present in primary tumor-derived exosomes, mainly involved in neuronal development, and 6 proteins in metastasis-derived exosomes related to cancer progression. Significant proteins obtain with statistical analysis performed between the two groups, reveal that primary tumor exosomes contain a higher level of proteins involved in extra-cellular matrix (ECM) assembly and adhesion, as well as in neuronal development. Exosomes isolated from bone-marrow metastasis exhibit proteins involved in ameboidal cell migration and mitochondrial activity. This work suggests that proteomic profiling of NB-derived exosomes reflects the tumor stage and may be considered as potential tumor biomarker.

Resistance to neoplastic transformation of ex-vivo expanded human mesenchymal stromal cells after exposure to supramaximal physical and chemical stress.

The risk of malignant transformation of ex-vivo expanded human mesenchymal stromal cells (huMSCs) has been debated in the last years; however, the biosafety of these cells after exposure to supramaximal physical and chemical stress has never been systematically investigated.We established an experimental in vitro model to induce supramaximal physical (ionizing radiation, IR) and chemical (starvation) stress on ex-vivo expanded bone marrow (BM)-derived huMSCs and investigated their propensity to undergo malignant transformation. To this aim, we examined MSC morphology, proliferative capacity, immune-phenotype, differentiation potential, immunomodulatory properties and genetic profile before and after stressor exposure. Furthermore, we investigated the cellular mechanisms underlying MSC response to stress. MSCs were isolated from 20 healthy BM donors and expanded in culture medium supplemented with 5% platelet lysate (PL) up to passage 2 (P2). At this stage, MSCs were exposed first to escalating doses of IR (30, 100, 200 Gy) and then to starvation culture conditions (1% PL).With escalating doses of radiation, MSCs lost their typical spindle-shaped morphology, their growth rate markedly decreased and eventually stopped (at P4-P6) by reaching early senescence. Irradiated and starved MSCs maintained their typical immune-phenotype, ability to differentiate into adipocytes/osteoblasts and to inhibit mitogen-induced T-cell proliferation. The study of the genetic profile of irradiated/starved MSCs did not show any alteration. While the induction of supramaximal stress triggered production of ROS and activation of DNA damage response pathway via multiple mechanisms, our data indicate that irradiated/starved MSCs, although presenting altered morphology/growth rate, do not display increased propensity for malignant transformation.

PCA3 in prostate cancer and tumor aggressiveness detection on 407 high-risk patients: a National Cancer Institute experience.

BACKGROUND: Prostate cancer (PCa) is the most common male cancer in Europe and the US. The early diagnosis relies on prostate specific antigen (PSA) serum test, even if it showed clear limits. Among the new tests currently under study, one of the most promising is the prostate cancer gene 3 (PCA3), a non-coding mRNA whose level increases up to 100 times in PCa tissues when compared to normal tissues. With the present study we contribute to the validation of the clinical utility of the PCA3 test and to the evaluation of its prognostic potential. METHODS: 407 Italian men, with two or more PCa risk factors and at least a previous negative biopsy, entering the Urology Unit of Regina Elena National Cancer Institute, were tested for PCA3, total PSA (tPSA) and free PSA (fPSA and f/tPSA) tests. Out of the 407 men enrolled, 195 were positive for PCa and 114 of them received an accurate staging with evaluation of the Gleason score (Gs). Then, the PCA3 score was correlated to biopsy outcome, and the diagnostic and prognostic utility were evaluated. RESULTS: Out of the 407 biopsies performed after the PCA3 test, 195 (48%) resulted positive for PCa; the PCA3 score was significantly higher in this population (p < 0.0001) differently to tPSA (p = 0.87). Moreover, the PCA3 test outperformed the f/tPSA (p = 0.01). The sensitivity (94.9) and specificity (60.1) of the PCA3 test showed a better balance for a threshold of 35 when compared to 20, even if the best result was achieved considering a cutoff of 51, with sensitivity and specificity of 82.1% and 79.3%, respectively. Finally, comparing values of the PCA3 test between two subgroups with increasing Gs (Gs ≤ 6 versus Gs ≥ 7) a significant association between PCA3 score and Gs was found (p = 0.02). CONCLUSIONS: The PCA3 test showed the best diagnostic performance when compared to tPSA and f/tPSA, facilitating the selection of high-risk patients that may benefit from the execution of a saturation prostatic biopsy. Moreover, the PCA3 test showed a prognostic value, as higher PCA3 score values are associated to a greater tumor aggressiveness.

Perioperative changes in pro and anticoagulant factors in prostate cancer patients undergoing laparoscopic and robotic radical prostatectomy with different anaesthetic techniques.

BACKGROUND: Laparoscopic prostatectomy (LRP) may activate clotting system influencing the risk of perioperative thrombosis in patients with prostate cancer. Moreover, different anaesthetic techniques can also modify coagulant factors. Thus, the aim of this study was to investigate the effects on pro- and anti-coagulant and fibrinolytic factors of two established types of anaesthesia in patients with prostate cancer undergoing elective LRP. METHODS: 102 patients with primary prostate cancer, who underwent conventional LRP or robot-assisted laparoscopic prostatectomy (RALP), were studied and divided into 2 groups to receive total intravenous anesthesia with target-controlled infusion (TIVA-TCI) or balanced inhalation anaesthesia (BAL) prior to surgery. Before the induction of anaesthesia (T0), 1 hr (T1) and 24 hrs post-surgery (T2), some pro-coagulant factors, fibronolysis markers, p-selectin and haemostatic system inhibitors were evaluated. RESULTS: Both TIVA-TCI and BAL patients showed a marked and significant increase in pro-coagulant factors and consequent reduction in haemostatic system inhibitors in the early post operative period (p ≤ 0.004 for each markers). Use of RALP showed a significant increase in prothrombotic markers as compared to LRP. In TIVA patients undergoing LRP, a significant reduction of p-selectin levels between T0 and T2 (p = 0.001) was observed as compared to BAL, suggesting a better protective effect on platelet activation of anaesthetic agents used for TIVA. CONCLUSIONS: Both anaesthetic techniques significantly seem to increase the risk of thrombosis in prostate cancer patients undergoing LRP, mainly when the robotic device was utilized, encouraging the use of a peri-operative thromboembolic prophylaxis in these patients.

Characterization of the prostate-specific antigen (PSA) catalytic mechanism: a pre-steady-state and steady-state study.

Prostate-specific antigen (PSA), an enzyme of 30 kDa grouped in the kallikrein family is synthesized to high levels by normal and malignant prostate epithelial cells. Therefore, it is the main biomarker currently used for early diagnosis of prostate cancer. Here, presteady-state and steady-state kinetics of the PSA-catalyzed hydrolysis of the fluorogenic substrate Mu-His-Ser-Ser-Lys-Leu-Gln-AMC (spanning from pH 6.5 to pH 9.0, at 37.0°C) are reported. Steady-state kinetics display at every pH value a peculiar feature, represented by an initial "burst" phase of the fluorescence signal before steady-state conditions are taking place. This behavior, which has been already observed in other members of the kallikrein family, suggests the occurrence of a proteolytic mechanism wherefore the acylation step is faster than the deacylation process. This feature allows to detect the acyl intermediate, where the newly formed C-terminal carboxylic acid of the cleaved substrate forms an ester bond with the -OH group of the Ser195 catalytic residue, whereas the AMC product has been already released. Therefore, the pH-dependence of the two enzymatic steps (i.e., acylation and deacylation) has been separately characterized, allowing the determination of pKa values. On this basis, possible residues are tentatively identified in PSA, which might regulate these two steps by interacting with the two portions of the substrate.

Focus on genetic and epigenetic events of colorectal cancer pathogenesis: implications for molecular diagnosis.

Originally, colorectal cancer (CRC) tumorigenesis was understood as a multistep process that involved accumulation of tumor suppressor genes and oncogenes mutations, such as APC, TP53 and KRAS. However, this assumption proposed a relatively limited repertoire of genetic alterations. In the last decade, there have been major advances in knowledge of multiple molecular pathways involved in CRC pathogenesis, particularly regarding cytogenetic and epigenetic events. Microsatellite instability, chromosomal instability and CpG island methylator phenotype are the most analyzed cytogenetic changes, while DNA methylation, modifications in histone proteins and microRNAs (miRNAs) were analyzed in the field of epigenetic alterations. Therefore, CRC development results from interactions at many levels between genetic and epigenetic amendments. Furthermore, hereditary cancer syndrome and individual or environmental risk factors should not be ignored. The difficulties in this setting are addressed to understand the molecular basis of individual susceptibility to CRC and to determine the roles of genetic and epigenetic alterations, in order to yield more effective prevention strategies in CRC patients and directing their treatment. This review summarizes the most investigated biomolecular pathways involved in CRC pathogenesis, their role as biomarkers for early CRC diagnosis and their possible use to stratify susceptible patients into appropriate screening or surveillance programs.

Critical appraisal of bevacizumab in the treatment of metastatic colorectal cancer.

Colorectal cancer is one of the most common cancers worldwide. The prognosis of patients with metastatic colorectal cancer in recent years has increased from 5 months with best supportive care to nearly 2 years with chemotherapy combined with bevacizumab, an antivascular endothelial growth factor monoclonal antibody. New prognostic and predictive biomarkers have been identified to guide chemotherapy in metastatic colorectal cancer, such as KRAS and BRAF oncogenes. However, the status of these oncogenes does not affect the efficacy of bevacizumab, and biomarkers predicting response to treatment with bevacizumab are still lacking. Addition of bevacizumab to regimens based on fluoropyrimidines or irinotecan has been shown to improve overall survival in treatment-naïve patients with metastatic colorectal cancer. Similarly, a significant increase in overall survival rate is achieved by adding bevacizumab to fluoropyrimidines and oxaliplatin in patients with disease progression. Bevacizumab has been found to be effective even when used as third-line therapy and later. In addition, cohort studies have shown that bevacizumab improves survival significantly despite disease progression. Finally, bevacizumab therapy in the neoadjuvant setting for the treatment of liver metastasis is well tolerated, safe, and effective.