Istituto Ortopedico Rizzoli

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is thatthere is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the completeprocess including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increasedautophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in manycases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as forreviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multipleassays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagyrelated protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

Bacteria were first detected in human tumors more than 100 years ago, but the characterization of the tumor microbiome has remained challenging because of its low biomass. We undertook a comprehensive analysis of the tumor microbiome, studying 1526 tumors and their adjacent normal tissues across seven cancer types, including breast, lung, ovary, pancreas, melanoma, bone, and brain tumors. We found that each tumor type has a distinct microbiome composition and that breast cancer has a particularly rich and diverse microbiome. The intratumor bacteria are mostly intracellular and are present in both cancer and immune cells. We also noted correlations between intratumor bacteria or their predicted functions with tumor types and subtypes, patients' smoking status, and the response to immunotherapy.

Soft tissue sarcomas (STSs) gather over 80 histological entities, with even more molecular subsets, characterised by a low to very low incidence in all populations. The majority of sarcomas arise from the soft tissue (close to 75%), with ∼15% gastrointestinal stromal tumours (GISTs) and 10% bone sarcomas. These ESMO–EURACAN (European Society for Medical Oncology–European Reference Network for rare adult solid cancers) Clinical Practice Guidelines cover STSs, while GISTs are covered by dedicated ESMO–EURACAN Clinical Practice Guidelines [1].

To the Editor: The reconstruction of large bone segments is an important clinical problem, and none of the approaches proposed thus far have proved very effective. In animals, repair and functional...

Of 327 patients who had a giant-cell tumor of bone and were seen at the Istituto Rizzoli, 293 were treated at the Institute, and 280 of these were followed for two to forty-four years. The distribution according to sex and age of the patient and site of the tumor was similar to the distributions in major reports of large series. The tumor usually involved the metaphysis and the epiphysis, but was occasionally limited to the metaphysis, and in only 2 per cent of the patients was it adjacent to an open growth plate. The tumor on occasion invaded the articular space, also involving the ligaments and the synovial membrane. Extension to an adjacent bone through the joint occurred in 5 per cent of the tumors. Our radiographic grading, which is roughly comparable with the staging system of Enneking et al., was Grade I in 4 per cent, II in 74 per cent, and III in 22 per cent of 266 patients before treatment. A pathological fracture was apparent on the first radiograph in 9 per cent of the patients. In the 280 patients with adequate follow-up, 331 surgical procedures were performed. The rate of local recurrence was 27 per cent in the 151 intralesional procedures, 8 per cent in the 122 marginal excisions, and zero in the fifty-eight wide or radical procedures. These results did not correlate with the radiographic grade of the lesion. Of the fifty-one local recurrences that were seen after treatment at our institution, 90 per cent appeared in the first three years after surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND: All living organisms are made of individual and identifiable cells, whose number, together with their size and type, ultimately defines the structure and functions of an organism. While the total cell number of lower organisms is often known, it has not yet been defined in higher organisms. In particular, the reported total cell number of a human being ranges between 10(12) and 10(16) and it is widely mentioned without a proper reference. AIM: To study and discuss the theoretical issue of the total number of cells that compose the standard human adult organism. SUBJECTS AND METHODS: A systematic calculation of the total cell number of the whole human body and of the single organs was carried out using bibliographical and/or mathematical approaches. RESULTS: A current estimation of human total cell number calculated for a variety of organs and cell types is presented. These partial data correspond to a total number of 3.72 × 10(13). CONCLUSIONS: Knowing the total cell number of the human body as well as of individual organs is important from a cultural, biological, medical and comparative modelling point of view. The presented cell count could be a starting point for a common effort to complete the total calculation.

INTRODUCTION: Administration of mesenchymal stem cells (MSCs) represents a promising treatment option for patients suffering from immunological and degenerative disorders. Accumulating evidence indicates that the healing effects of MSCs are mainly related to unique paracrine properties, opening opportunities for secretome-based therapies. Apart from soluble factors, MSCs release functional small RNAs via extracellular vesicles (EVs) that seem to convey essential features of MSCs. Here we set out to characterize the full small RNAome of MSC-produced exosomes. METHODS: We set up a protocol for isolating exosomes released by early passage adipose- (ASC) and bone marrow-MSCs (BMSC) and characterized them via electron microscopy, protein analysis and small RNA-sequencing. We developed a bioinformatics pipeline to define the exosome-enclosed RNA species and performed the first complete small RNA characterization of BMSCs and ASCs and their corresponding exosomes in biological replicates. RESULTS: Our analysis revealed that primary ASCs and BMSCs have highly similar small RNA expression profiles dominated by miRNAs and snoRNAs (together 64-71 %), of which 150-200 miRNAs are present at physiological levels. In contrast, the miRNA pool in MSC exosomes is only 2-5 % of the total small RNAome and is dominated by a minor subset of miRNAs. Nevertheless, the miRNAs in exosomes do not merely reflect the cellular content and a defined set of miRNAs are overrepresented in exosomes compared to the cell of origin. Moreover, multiple highly expressed miRNAs are precluded from exosomal sorting, consistent with the notion that these miRNAs are involved in functional repression of RNA targets. While ASC and BMSC exosomes are similar in RNA class distribution and composition, we observed striking differences in the sorting of evolutionary conserved tRNA species that seems associated with the differentiation status of MSCs, as defined by Sox2, POU5F1A/B and Nanog expression. CONCLUSIONS: We demonstrate that primary MSCs release small RNAs via exosomes, which are increasingly implicated in intercellular communications. tRNAs species, and in particular tRNA halves, are preferentially released and their specific sorting into exosomes is related to MSC tissue origin and stemness. These findings may help to understand how MSCs impact neighboring or distant cells with possible consequences for their therapeutic usage.

A significant proportion of medical implants become the focus of a device-related infection, difficult to eradicate because bacteria that cause these infections live in well-developed biofilms. Biofilm is a microbial derived sessile community characterized by cells that are irreversibly attached to a substratum or interface to each other, embedded in a matrix of extracellular polymeric substances that they have produced. Bacterial adherence and biofilm production proceed in two steps: first, an attachment to a surface and, second, a cell-to-cell adhesion, with pluristratification of bacteria onto the artificial surface. The first step requires the mediation of bacterial surface proteins, the cardinal of which is similar to S. aureus autolysin and is denominated AtlE. In staphylococci the matrix of extracellular polymeric substances of biofilm is a polymer of beta-1,6-linked N-acetylglucosamine (PIA), whose synthesis is mediated by the ica operon. Biofilm formation is partially controlled by quorum sensing, an interbacterial communication mechanism dependent on population density. The principal implants that can be compromised by biofilm associated infections are: central venous catheters, heart valves, ventricular assist devices, coronary stents, neurosurgical ventricular shunts, implantable neurological stimulators, arthro-prostheses, fracture-fixation devices, inflatable penile implants, breast implants, cochlear implants, intraocular lenses, dental implants. Biofilms play an important role in the spread of antibiotic resistance. Within the high dense bacterial population, efficient horizontal transfer of resistance and virulence genes takes place. In the future, treatments that inhibit the transcription of biofilm controlling genes might be a successful strategy in inhibiting these infections.A significant proportion of medical implants become the focus of a device-related infection, difficult to eradicate because bacteria that cause these infections live in well-developed biofilms. Biofilm is a microbial derived sessile community characterized by cells that are irreversibly attached to a substratum or interface to each other, embedded in a matrix of extracellular polymeric substances that they have produced. Bacterial adherence and biofilm production proceed in two steps: first, an attachment to a surface and, second, a cell-to-cell adhesion, with pluristratification of bacteria onto the artificial surface. The first step requires the mediation of bacterial surface proteins, the cardinal of which is similar to S. aureus autolysin and is denominated AtlE. In staphylococci the matrix of extracellular polymeric substances of biofilm is a polymer of beta-1,6-linked N-acetylglucosamine (PIA), whose synthesis is mediated by the ica operon. Biofilm formation is partially controlled by quorum sensing, an interbacterial communication mechanism dependent on population density. The principal implants that can be compromised by biofilm associated infections are: central venous catheters, heart valves, ventricular assist devices, coronary stents, neurosurgical ventricular shunts, implantable neurological stimulators, arthro-prostheses, fracture-fixation devices, inflatable penile implants, breast implants, cochlear implants, intra-ocular lenses, dental implants. Biofilms play an important role in the spread of antibiotic resistance. Within the high dense bacterial population, efficient horizontal transfer of resistance and virulence genes takes place. In the future, treatments that inhibit the transcription of biofilm controlling genes might be a successful strategy in inhibiting these infections.

Gastrointestinal stromal tumours (GISTs) are rare tumours, with an estimated unadjusted incidence of around 1/100 000/year [1.Nilsson B. Bümming P. Meis-Kindblom J.M. et al.Gastrointestinal stromal tumors: the incidence, prevalence, clinical course, and prognostication in the preimatinib mesylate era–a population-based study in western Sweden.Cancer. 2005; 103: 821-829Crossref PubMed Scopus (1027) Google Scholar]. This only covers clinically relevant GISTs, since, if investigated, a much higher number of lesions ≤ 1 cm in diameter (microGISTs) can be found at histopathological examination of stomach tissue in middle-aged and elderly individuals. There is a slight prevalence in males. The median age is around 60–65 years, with a wide range. Occurrence in children is very rare. Paediatric GIST represents a clinically and molecularly distinct subset, marked by female predominance, absence of KIT/platelet-derived growth factor alpha (PDGFRA) mutations, frequent mutations or silencing of the four genes that encode the subunits of the succinate dehydrogenase (SDH) enzyme complex, gastric multicentric location and possible lymph node metastases [2.Pappo A.S. Janeway K.A. Pediatric gastrointestinal stromal tumors.Hematol Oncol Clin North Am. 2009; 23 (vii): 15-34Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar]. Some syndromes are linked to GISTs: •The Carney triad syndrome, marked by gastric GISTs, paraganglioma and pulmonary chondromas (these may occur at different ages) [3.Zhang L. Smyrk T.C. Young Jr, W.F. et al.Gastric stromal tumors in Carney triad are different clinically, pathologically, and behaviorally from sporadic gastric gastrointestinal stromal tumors: findings in 104 cases.Am J Surg Pathol. 2010; 34: 53-64Crossref PubMed Scopus (167) Google Scholar];•Carney–Stratakis syndrome, marked by a dyad of GIST and paraganglioma [4.Gaal J. Stratakis C.A. Carney J.A. et al.SDHB immunohistochemistry: a useful tool in the diagnosis of Carney–Stratakis and Carney triad gastrointestinal stromal tumors.Mod Pathol. 2011; 24: 147-151Crossref PubMed Scopus (162) Google Scholar, 5.Pasini B. McWhinney S.R. Bei T. et al.Clinical and molecular genetics of patients with the Carney–Stratakis syndrome and germline mutations of the genes coding for the succinate dehydrogenase subunits SDHB, SDHC, and SDHD.Eur J Hum Genet. 2008; 16: 79-88Crossref PubMed Scopus (363) Google Scholar]; and•Neurofibromatosis type 1(NF1), possibly leading to wild-type (WT), often multicentric GIST, predominantly located in the small bowel [6.Miettinen M. Fetsch J.F. Sobin L.H. Lasota J. Gastrointestinal stromal tumors in patients with neurofibromatosis 1: a clinicopathologic and molecular genetic study of 45 cases.Am J Surg Pathol. 2006; 30: 90-96Crossref PubMed Scopus (377) Google Scholar]. Families with germline autosomal dominant mutations of KIT are an extremely rare finding, presenting with multiple GISTs at an early age, possibly along with other associated features such as pigmented skin macules, urticaria pigmentosa and diffuse hyperplasia of the interstitial cells of Cajal in the gut wall. When small oesophagogastric or duodenal nodules < 2 cm in size are detected, endoscopic biopsy may be difficult and laparoscopic/laparotomic excision may be the only way to make a histological diagnosis. Many of these small nodules, if diagnosed as GISTs, will be either low-risk or entities whose clinical significance remains unclear. Therefore, the standard approach to patients with oesophagogastric or duodenal nodules < 2 cm is endoscopic ultrasound assessment and then follow-up, reserving excision for patients whose tumour increases in size or becomes symptomatic [IV, C]. As an option, the patient can choose to undergo a histological assessment, also depending on age, life expectancy and comorbidities. If follow-up is the choice, an evidence-based, optimal surveillance policy is lacking. A logical approach may be to have a short-term first control (e.g. at 3 months) and then, in the case of no evidence of growth, a more relaxed follow-up schedule may be selected. In a histologically proven small GIST, standard treatment is excision, unless major morbidity is expected. Alternatively, in the case of a likely low-risk GIST on biopsy, the decision can be made with the patient to follow up the lesion. However, an exception is the standard approach to rectal nodules represented by biopsy or excision after endorectal ultrasound assessment and pelvic magnetic resonance imaging (MRI), regardless of the tumour size and mitotic rate. In fact, the progression risk of a clinically significant GIST at this site is higher, its prognosis is significantly worse compared with most gastric GISTs and the local implications for surgery are more critical. A follow-up policy may be an option, to be discussed with the patient, in the case of small lesions and whenever the surgical risk is particularly high (comorbidities, age, etc.). The standard approach to tumours ≥2 cm in size is biopsy/excision, because they are associated with a higher risk of progression if confirmed as GIST [IV, C]. If there is an abdominal nodule not amenable to endoscopic assessment, laparoscopic/laparotomic excision is the standard approach. If there is a mass, especially if surgery is likely to be a multivisceral resection, multiple core needle biopsies are the standard approach. They should be obtained through endoscopic ultrasound guidance, or through an ultrasound/computed tomography (CT)-guided percutaneous approach. This may allow the surgeon to plan the best approach according to the histological diagnosis and avoid surgery for diseases which might not benefit (e.g. lymphomas, mesenteric fibromatosis and germ cell tumours). The risk of peritoneal contamination is negligible if the procedure is properly carried out. Moreover, lesions at risk in this regard (e.g. cystic masses) should be biopsied only in specialised centres. Immediate laparoscopic/laparotomic excision is an option on an individualised basis, especially if surgery is limited. If a patient presents with obvious metastatic disease, a biopsy of the metastatic focus is sufficient and the patient usually does not require a laparotomy for diagnostic purposes. The tumour sample should be fixed in 4% buffered formalin (Bouin fixative should not be used, since it prevents molecular analysis). Pathologically, the diagnosis of GIST relies on morphology and immunohistochemistry, the latter being positive for CD117 (KIT) and/or DOG1 (see Table 1) [7.Rubin B.P. Blanke C.D. Demetri G.D. et al.Protocol for the examination of specimens from patients with gastrointestinal stromal tumor.Arch Pathol Lab Med. 2010; 134: 165-170Crossref PubMed Google Scholar, 8.Novelli M. Rossi S. Rodriguez-Justo M. et al.DOG1 and CD117 are the antibodies of choice in the diagnosis of gastrointestinal stromal tumours.Histopathology. 2010; 57: 259-270Crossref PubMed Scopus (147) Google Scholar]. A proportion of GISTs (in the range of 5%) are CD117-negative. The mitotic count has a prognostic value and should be expressed as the number of mitoses on a total area of 5 mm2 [which replaces the former 50 high-power field (HPF) area]. Mutational analysis for known mutations involving KIT and PDGFRA can confirm the diagnosis of GIST, if doubtful (particularly in rare CD117/DOG1-negative suspect GIST). Mutational analysis has a predictive value for sensitivity to molecular-targeted therapy and to prognostic value. Its inclusion in the diagnostic work-up of all GISTs should be considered standard practice [II, A] (with the possible exclusion of < 2 cm non-rectal GISTs, which are very unlikely ever to be candidates for medical treatment). Centralisation of mutational analysis in a laboratory enrolled in an external quality assurance programme and with expertise in the may be diagnosis is more for GIST, to confirm the diagnosis of GIST with an at a In GIST, for is to In GIST an syndrome should be Rossi S. M. et GIST is a for neurofibromatosis type 1 PubMed Scopus Google Scholar]. The of tissue is to allow molecular to be made at a for tumour to local and should be and for medical treatment or for medical treatment for medical treatment of of growth factor succinate in a of of growth factor succinate treatment is involving medical as as as should be carried in for and GISTs and/or expertise and a high number of patients The for node and of tumours the prognostic in GIST (see Table of for GIST from of of of to of KIT site in KIT or PDGFRA of and gastrointestinal stromal neurofibromatosis type growth factor tumour for from of with from in a GIST, gastrointestinal stromal neurofibromatosis type growth factor tumour for from of with from are the mitotic tumour size and tumour site GISTs have a prognosis small bowel or rectal is an prognostic factor and should be regardless of it or Mutational has not in risk at have a distinct GISTs have clinical and GIST with are associated with a prognosis and to risk have A risk by the of which the mitotic tumour size and tumour the prognostic in GISTs M. Lasota J. Gastrointestinal stromal tumors: on molecular and Pathol Lab Med. 2006; PubMed Google Scholar, M. Lasota J. Gastrointestinal stromal tumors: and prognosis at different Pathol. 2006; PubMed Scopus Google Scholar]. A all has on M. et and of a prognostic for after surgical of gastrointestinal stromal a 2009; Full Text Full Text PDF PubMed Scopus Google Scholar]. When these it is to that the mitotic and tumour size are that are through a of of GIST patients not with which the mitotic and tumour size as tumour is considered in to tumour site J. et of of gastrointestinal stromal tumour after an analysis of population-based Full Text Full Text PDF PubMed Scopus Google Scholar]. They have a that most the and the abdominal and pelvic is the of choice for and may be an rectal GISTs, and laboratory the work-up of the The of an tomography or is useful early of the tumour to molecular-targeted therapy is of The standard treatment of GISTs is surgical excision of the with no of clinically lymph If excision is the to follow the of surgery A] et of and surgery for gastric gastrointestinal stromal Google Scholar]. A approach is in patients have tumours, because of the risk of tumour which is associated with a very high risk of excision is the an excision whose are of tumour When surgery major and medical treatment is not the decision can be made with the patient to possible [IV, This is more for low-risk the of that surgery is associated with a worse If excision carried may be an option, the site of can be and major are not The risk of can be as by risk treatment with for 3 associated with a and in with 1 of therapy in patients in a M. et for stromal analysis of a Clin 34: PubMed Scopus (147) Google Scholar]. a that for a of 1 can in GISTs a diameter 3 cm with a et mesylate after of gastrointestinal stromal a 2009; Full Text Full Text PDF PubMed Scopus Google Scholar]. Therefore, therapy with for 3 is the standard treatment for patients with a significant risk of A is the risk is T. et treatment of GIST with or A on of the and the the the on GIST, the and the J 2009; Full Text Full Text PDF PubMed Scopus Google Scholar]. clinical are to of therapy in The benefit associated of may according to the type of being in patients with KIT mutations et of KIT and PDGFRA mutations on in patients with gastrointestinal stromal tumors with an analysis of a clinical PubMed Scopus Google Scholar, et and molecular features with after therapy of stromal the Clin PubMed Scopus Google Scholar]. Mutational analysis is to make a clinical decision There is a that PDGFRA GISTs should not be with the of sensitivity of this in and in [IV, the the of a higher of in the case of an KIT in GIST, to this in the for this [II, M. et mutations and for in patients with gastrointestinal stromal J 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, et of and clinical in the North of mesylate for treatment of gastrointestinal stromal by and and Clin 2008; PubMed Scopus Google Scholar, Demetri G.D. et mutations and in patients with metastatic gastrointestinal stromal Clin PubMed Scopus Google Scholar]. may this which is not in the by regard to GIST, there is a on treatment in and GISTs [IV, This of sensitivity to and other in the as as which is often more of to sufficient and be to best in the rare In case of tumour at the of there is of tumour cells the peritoneal peritoneal can be to This the patient at a very high risk of peritoneal P. et of in patients with gastrointestinal stromal J 2010; PubMed Scopus Google Scholar]. Therefore, these patients should be considered for therapy [IV, The optimal of treatment in these is the these cells should be considered as If surgery is not or it be through surgery in the case of total and all other major with is standard A] J. Blanke C.D. et of mesylate for and gastrointestinal stromal early of Surg 2009; PubMed Scopus Google Scholar, P. P. et in gastrointestinal stromal tumors the Surg PubMed Scopus Google Scholar]. This may also be the case if the surgeon that the surgical is after (e.g. the risk of and tumour is A may be the of mitotic for risk for A biopsy with histological and mutational is to confirm the histological to to therapy with (e.g. PDGFRA and to the for KIT tumour after surgery is carried out. tumour assessment is to avoid surgery in the case of imaging it possible to the tumour very a particularly in the absence of mutational There are to the on to treatment it can be a or surgery and can be the patient from is the standard treatment for and metastatic A] C.D. Demetri G.D. M. et from a of mesylate for patients with or metastatic gastrointestinal stromal tumors Clin 2008; PubMed Scopus Google Scholar, C.D. Demetri G.D. et mesylate at in patients with or metastatic gastrointestinal stromal tumors the Clin 2008; PubMed Scopus Google Scholar, J. J. et in gastrointestinal stromal tumours with Full Text Full Text PDF PubMed Scopus Google Scholar, J. et of patients with stromal tumours to a of after progression on J 2005; Full Text Full Text PDF PubMed Scopus Google as as for patients with not is also the standard treatment for patients with metastatic have all lesions surgery is not as a approach in the metastatic The standard of is However, have that patients with tumours the KIT have significantly on a higher which is as standard treatment in this of of for the treatment of or metastatic gastrointestinal stromal tumors: a of Clin 2010; PubMed Scopus Google Scholar]. with a PDGFRA are to S. et al.Clinical and treatment in a of PDGFRA gastrointestinal stromal tumour J Full Text Full Text PDF PubMed Scopus Google and other and candidates for clinical on this is doubtful patients with GIST benefit from there are of of K.A. et treatment in patients with GIST of 2009; PubMed Scopus Google Scholar]. In the metastatic treatment with should be since treatment is by tumour lesions have A] et of in patients with gastrointestinal stromal tumours after 3 of an 3 2010; Full Text Full Text PDF PubMed Scopus Google Scholar]. When treatment is the patient should be to the of with as as with and and the best to should be by of and a policy of and should be in the case of that of are associated with a worse a with the has G.D. et are with clinical benefit in patients with gastrointestinal stromal Clin 2009; PubMed Scopus Google Scholar]. from its to the assessment of may be useful in the case patients that at a risk of major or patients with surgical to and progression on to the to the to of the tumour should be carried in the early of should be the since the risk of progression excision of metastatic has to be associated with a the patient is to it has this is to surgery or to patient J. et surgery in is it in all 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. et of gastrointestinal stromal tumors after treatment with therapy Clin 2006; 24: PubMed Scopus Google Scholar, Blanke C.D. et of mesylate for and gastrointestinal stromal tumors: follow-up of Surg PubMed Scopus Google Scholar, S. P. P. et follow-up of patients with GIST in the of analysis of prognostic J Surg Full Text Full Text PDF PubMed Scopus Google Scholar]. not early because of for a small positive in which all patients peritoneal et there a of surgery in patients with or metastatic gastrointestinal stromal tumours to a in J Full Text Full Text PDF PubMed Scopus Google Scholar]. the surgical option should be individualised after the decision with the patient in the case of C]. excision of has not in surgery of such as the a has associated with a in the range as for treatment with Therefore, this may be a option for an patient with [IV, C]. (e.g. local such as or may be selected. In the case of tumour progression on an option may be to the to with the exception of mutations with the C.D. Demetri G.D. M. et from a of mesylate for patients with or metastatic gastrointestinal stromal tumors Clin 2008; PubMed Scopus Google Scholar, C.D. Demetri G.D. et mesylate at in patients with or metastatic gastrointestinal stromal tumors the Clin 2008; PubMed Scopus Google Scholar, J. J. et in gastrointestinal stromal tumours with Full Text Full Text PDF PubMed Scopus Google Scholar, J. et of patients with stromal tumours to a of after progression on J 2005; Full Text Full Text PDF PubMed Scopus Google Scholar]. is particularly useful in the case of a KIT GIST a higher not from the possibly in the case of in or in the case of molecular progression on imaging should be to the (see patient should be as a possible of tumour as as with In the case of confirmed progression or rare on to through and possibly standard treatment is A] G.D. et and of in patients with gastrointestinal stromal tumour after of a 2006; Full Text Full Text PDF PubMed Scopus Google Scholar]. The in of a have that a with a is and no has carried a clinical S. et al.Clinical of of in patients with gastrointestinal stromal tumour after J 2009; Full Text Full Text PDF PubMed Scopus Google Scholar]. This schedule be considered an option on an individualised C]. confirmed progression on a that at the of for 3 of can significantly This as it becomes is standard therapy for patients on or to to and A] G.D. P. et and of for gastrointestinal stromal tumours after of and an 3 Full Text Full Text PDF PubMed Scopus Google Scholar]. with a metastatic GIST should be considered for in clinical of or There is evidence that patients have on may benefit with the et of to control metastatic or gastrointestinal stromal tumours after of and a 3 Full Text Full Text PDF PubMed Scopus Google Scholar]. there is evidence that a treatment with a in the case of disease, may progression as to it no other option is at the at in a proportion of patients with a Therefore, or of treatment progression with to which the patient has is an option [II, the other the of of of clinical should be because of the for have in in with of in only a of total is complex, and early in should be confirmed by an tumour in most patients may only in tumour on or these may tumour in tumour should be considered as the tumour an in the tumour size may be of the tumour if the tumour on is et of tomography and tomography in patients with metastatic gastrointestinal stromal at a with of tomography Clin PubMed Scopus Google Scholar]. The of lesions also be to the of Therefore, tumour size and tumour on or in or should be considered as for tumour has proven to be in early assessment of tumour and may be useful in there is or early of the is particularly useful (e.g. S. et of in gastrointestinal stromal patients with Med. PubMed Scopus Google Scholar]. However, a small proportion of GISTs have no The absence of tumour progression after of treatment is also considered as tumour M. J. et of progression as by in tumors in stromal tumors with the Clin 2009; PubMed Scopus Google Scholar]. the other tumour progression may not be by in the tumour In fact, in the tumour tumour lesions may be of tumour A progression is the the by which a of a becomes S. J. et al.Gastrointestinal stromal of after to 2005; PubMed Scopus Google Scholar]. There are no to the optimal follow-up policy of patients with occur more often to the and/or of lesions and other may be rare along the of metastatic with of The mitotic likely the at which assessment on the mitotic tumour size and tumour site may be useful in the follow-up patients have a from the of patients may have a this is much follow-up The optimal follow-up are not As an in patients undergo a follow-up with an abdominal or for 3 therapy (with a clinical follow-up to the to the of unless then on of therapy 3 for 2 years, then 5 from and for an 5 low-risk tumours, the of a follow-up is not if this may be carried with abdominal or for 5 low-risk GISTs not require follow-up, the risk is not is a factor to especially in low-risk GIST, with abdominal being an L. P. et of a follow-up in patients by gastrointestinal stromal J Full Text Full Text PDF PubMed Scopus Google Scholar]. have by in with the for rare in with the standard for They are to the standard approach to treatment and on and are to to the according to the of are represented by the of the and by all to the of considered to be are as may be to the patient as for a decision in of as as evidence not is the the of disease, and are to the the The relevant has by the A of is in Table of evidence and of have the in Table considered standard clinical practice by the of and ultrasound assessment and then follow-up is the standard approach for patients with oesophagogastric or duodenal nodules < 2 cm [IV, is the standard approach to tumours 2 cm in size [IV, analysis inclusion in the diagnostic work-up of all GISTs should be considered standard practice [II, A] (with the possible exclusion of < 2 cm non-rectal of standard treatment of GISTs is surgical excision of the with no of clinically lymph excision is the to follow the of surgery surgery major and medical treatment is not the decision can be made with the patient to possible [IV, therapy with for 3 is the standard treatment of patients with a significant risk of GISTs should not be with [IV, treatment should be in and GISTs [IV, at a very high risk of peritoneal (in case of tumour at the of should be considered for therapy [IV, surgery with no major is not with is standard of is the standard treatment of and metastatic is also the standard treatment for patients with metastatic have all lesions surgery is not as a approach in the metastatic The standard of is treatment of patients with KIT is of the metastatic treatment with should be unless or patient to of metastatic should be individualised after the decision with the patient in the case of excision of should be considered for an patient with [IV, the case of tumour progression on of the can be to (with the exception of the case of confirmed progression or rare on standard treatment is at the of for 3 of is the standard therapy for patients on or to to and or of treatment progression with to which the patient has is an option [II, gastrointestinal stromal neurofibromatosis type growth factor no succinate in a Table of evidence and of from the of of the of of from at of quality for or of or with a of or of such or of with or control case of evidence for with a clinical or evidence for with a clinical evidence for or benefit does not the risk or the evidence or for not evidence or for of the of C.A. of the for cell PubMed Scopus Google Scholar]. in a GIST, gastrointestinal stromal neurofibromatosis type growth factor no succinate has for from and by has from has or from and has for and and by and has and from and and from has from and has for and has from and and and from and has for and being a of the for and from and is a of the for and has for and has by has in for and has by has and from and has from and has from and has for from and from and and from and from and has from and has with in with and has from and to clinical has by and and has and from and has with and in and is a for and has from for has being a for for the of a tumour has from and and for has from and has from and has on for and and has from has on for and and has from and has for from and has as a of for has from has or for is a of of has from and has from has from and from and from and has for and has from has and/or from and has and from and has from and for or and have no of and have not of to stromal for treatment and of Oncol PDF

Dysregulated signaling through the Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways is often the result of genetic alterations in critical components in these pathways or upstream activators. Unrestricted cellular proliferation and decreased sensitivity to apoptotic-inducing agents are typically associated with activation of these pro-survival pathways. This review discusses the functions these pathways have in normal and neoplastic tissue growth and how they contribute to resistance to apoptotic stimuli. Crosstalk and commonly identified mutations that occur within these pathways that contribute to abnormal activation and cancer growth will also be addressed. Finally the recently described roles of these pathways in cancer stem cells, cellular senescence and aging will be evaluated. Controlling the expression of these pathways could ameliorate human health.

The ability of marrow-derived osteoprogenitor cells to promote repair of critical-size tibial gaps upon autologous transplantation on a hydroxyapatite ceramic (HAC) carrier was tested in a sheep model. Conditions for in vitro expansion of sheep bone marrow stromal cells (BMSC) were established and the osteogenic potential of the expanded cells was validated. Ectopic implantation of sheep BMSC in immunocompromised mice led to extensive bone formation. When used to repair tibial gaps in sheep, cell-loaded implants (n = 2) conducted a far more extensive bone formation than did cell-free HAC cylinders (n = 2) over a 2-month period. In cell-loaded implants, bone formation was found to occur both within the internal macropore space and around the HAC cylinder while in control cell-free implants, bone formation was limited mostly to the outer surface and was not observed in most of the inner pores. As tested in an indentation assay, the stiffness of the complex HAC-bone material was found to be higher in cell-loaded implants compared to controls. Our pilot study on a limited number of large-sized animals suggests that the use of autologous BMSC in conjunction with HAC-based carriers results in faster bone repair compared to HAC alone. Potentially this combination could be used clinically in the treatment of extensive long bone defects.

BACKGROUND: Massive endoprostheses provide orthopaedic oncologists with many reconstructive options after tumor resection, although failure rates are high. Because the number of these procedures is limited, failure of these devices has not been studied or classified adequately. This investigation is a multicenter review of the use of segmental endoprostheses with a focus on the modes, frequency, and timing of failure. METHODS: Retrospective reviews of the operative databases of five institutions identified 2174 skeletally mature patients who received a large endoprosthesis for tumor resection. Patients who had failure of the endoprosthesis were identified, and the etiology and timing of failure were noted. Similar failures were tabulated and classified on the basis of the risk of amputation and urgency of treatment. Statistical analysis was performed to identify dependent relationships among mode of failure, anatomic location, and failure timing. A literature review was performed, and similar analyses were done for these data. RESULTS: Five hundred and thirty-four failures were identified. Five modes of failure were identified and classified: soft-tissue failures (Type 1), aseptic loosening (Type 2), structural failures (Type 3), infection (Type 4), and tumor progression (Type 5). The most common mode of failure in this series was infection; in the literature, it was aseptic loosening. Statistical dependence was found between anatomic location and mode of failure and between mode of failure and time to failure. Significant differences were found in the incidence of failure mode Types 1, 2, 3, and 4 when polyaxial and uniaxial joints were compared. Significant dependence was also found between failure mode and anatomic location in the literature data. CONCLUSIONS: There are five primary modes of endoprosthetic failure, and their relative incidences are significantly different and dependent on anatomic location. Mode of failure and time to failure also show a significant dependence. Because of these relationships, cumulative reporting of segmental failures should be avoided because anatomy-specific trends will be missed. Endoprosthetic design improvements should address failure modes specific to the anatomic location.

The perspectives of regenerative medicine are still severely hampered by the host response to biomaterial implantation, despite the robustness of technologies that hold the promise to recover the functionality of damaged organs and tissues. In this scenario, the cellular and molecular events that decide on implant success and tissue regeneration are played at the interface between the foreign body and the host inflammation, determined by innate and adaptive immune responses. To avoid adverse events, rather than the use of inert scaffolds, current state of the art points to the use of immunomodulatory biomaterials and their knowledge-based use to reduce neutrophil activation, and optimize M1 to M2 macrophage polarization, Th1 to Th2 lymphocyte switch, and Treg induction. Despite the fact that the field is still evolving and much remains to be accomplished, recent research breakthroughs have provided a broader insight on the correct choice of biomaterial physicochemical modifications to tune the reaction of the host immune system to implanted biomaterial and to favor integration and healing.

Primary bone tumours are rare, accounting for < 0.2% of malignant neoplasms registered in the EUROCARE (European Cancer Registry based study on survival and care of cancer patients) database [1]. Different bone tumour subtypes have distinct patterns of incidence, and each has no more than 0.3 incident cases per 100 000 per year. Osteosarcoma (OS) and Ewing sarcoma (ES) have a relatively high incidence in the second decade of life, whereas chondrosarcoma (CS) is more common in older age [2–4].

The application of massively parallel sequencing technology to the field of skeletal disorders has boosted the discovery of the underlying genetic defect for many of these diseases. It has also resulted in the delineation of new clinical entities and the identification of genes and pathways that had not previously been associated with skeletal disorders. These rapid advances have prompted the Nosology Committee of the International Skeletal Dysplasia Society to revise and update the last (2015) version of the Nosology and Classification of Genetic Skeletal Disorders. This newest and tenth version of the Nosology comprises 461 different diseases that are classified into 42 groups based on their clinical, radiographic, and/or molecular phenotypes. Remarkably, pathogenic variants affecting 437 different genes have been found in 425/461 (92%) of these disorders. By providing a reference list of recognized entities and their causal genes, the Nosology should help clinicians achieve accurate diagnoses for their patients and help scientists advance research in skeletal biology.

Extensive bone loss is still a major problem in orthopedics. A number of different therapeutic approaches have been developed and proposed, but so far none have proven to be fully satisfactory. We used a new tissue engineering approach to treat four patients with large bone diaphysis defects and poor therapeutic alternatives. To obtain implantable three-dimensional (3D) living constructs, cells isolated from the patients' bone marrow stroma were expanded in culture and seeded onto porous hydroxyapatite (HA) ceramic scaffolds designed to match the bone deficit in terms of size and shape. During the surgical session, an Ilizarov apparatus or a monoaxial external fixator was positioned on the patient's affected limb and the ceramic cylinder seeded with cells was placed in the bone defect. Patients were evaluated at different postsurgery time intervals by conventional radiographs and computed tomography (CT) scans. In one patient, an angiographic evaluation was performed at 6.5 years follow-up. In this study we analyze the long-term outcome of these patients following therapy. No major complications occurred in the early or late postoperative periods, nor were major complaints reported by the patients. No signs of pain, swelling, or infection were observed at the implantation site. Complete fusion between the implant and the host bone occurred 5 to 7 months after surgery. In all patients at the last follow-up (6 to 7 years postsurgery in patients 1 to 3), a good integration of the implants was maintained. No late fractures in the implant zone were observed. The present study shows the long-term durability of bone regeneration achieved by a bone engineering approach. We consider the obtained results very promising and propose the use of culture-expanded osteoprogenitor cells in conjunction with porous bioceramics as a real and significant improvement in the repair of critical-sized long bone defects.

PURPOSE: The aim of our study is to compare the efficacy of platelet-rich plasma (PRP) and viscosupplementation (hyaluronic acid [HA]) intra-articular injections for the treatment of knee cartilage degenerative lesions and osteoarthritis (OA). METHODS: The study involved 150 patients affected by cartilage degenerative lesions and early and severe OA. Fifty symptomatic patients were treated with 3 autologous PRP intra-articular injections and were evaluated prospectively at enrollment and at 2- and 6-month follow-up. The results obtained were compared with 2 homogeneous groups of patients treated with HA injections. One group was treated with injections of high-molecular weight HA; the other group was treated with low-molecular weight (LW) HA. International Knee Documentation Committee and EQ VAS scores were used for clinical evaluation; adverse events and patient satisfaction were also recorded. RESULTS: At 2 months' follow-up, the PRP and LW HA groups showed a similar improvement, with higher results compared with the high-molecular weight HA group (P < .005). At 6 months' follow-up, better results were observed in the PRP group (P < .005). PRP and LW HA treatments offered similar results in patients aged over 50 years and in the treatment of advanced OA. PRP showed a better performance compared with HA in younger patients affected by cartilage lesions or early OA. CONCLUSIONS: Autologous PRP injections showed more and longer efficacy than HA injections in reducing pain and symptoms and recovering articular function. Better results were achieved in younger and more active patients with a low degree of cartilage degeneration, whereas a worse outcome was obtained in more degenerated joints and in older patients, in whom results similar to those of viscosupplementation have been observed. LEVEL OF EVIDENCE: Level II, prospective comparative study.

William H. Chappell 1 , Linda S. Steelman 1,2 , Jacquelyn M. Long 2 , Ruth C. Kempf 2 , Stephen L. Abrams 1 , Richard A. Franklin 1 , J&ouml;rg B&auml;secke 3 , Franca Stivala 4 , Marco Donia 4 , Paolo Fagone 4 , Graziella Malaponte 4 , Maria C. Mazzarino 4 , Ferdinando Nicoletti 4 , Massimo Libra 4 , Danijela Maksimovic-Ivanic 5 , Sanja Mijatovic 5 , Giuseppe Montalto 6 , Melchiorre Cervello 7 , Piotr Laidler 8 , Michele Milella 9 , Agostino Tafuri 10 , Antonio Bonati 11 , Camilla Evangelisti 12 , Lucio Cocco 12 , Alberto M. Martelli 12,13 , and James A. McCubrey 1 1 Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University 2 Department of Physics, Greenville, NC 27858 USA 3 Department of Medicine University of G&ouml;ttingen, G&ouml;ttingen, Germany 4 Department of Biomedical Sciences, University of Catania, Catania, Italy 5 Department of Immunology, Institute for Biological Research &ldquo;Sinisa Stankovic&rdquo;, University of Belgrade, Belgrade, Serbia 6 Department of Internal Medicine and Specialties, University of Palermo, Palermo, Italy 7 Consiglio Nazionale delle Ricerche, Istituto di Biomedicina e Immunologia Molecolare &ldquo;Alberto Monroy&rdquo;, Palermo, Italy 8 Department of Medical Biochemistry Jagiellonian University Medical College, Krakow, Poland 9 Regina Elena Cancer Center, Via Elio Chianesi n.53, Rome 00144, Italy 10 University of Rome, La Sapienza, Department of Hematology-Oncology, Via Benevento 6, Rome 99161, Italy 11 University Hospital of Parma, Unit of Hematology and Bone-Marrow Transplantation, Via Gramsi n.14, Parma 43100, Italy 12 Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell&rsquo;Apparato Locomotore, Universit&agrave; di Bologna, Bologna, Italy 13 IGM-CNR, Sezione di Bologna, C/o IOR, Bologna, Italy Keywords: Targeted Therapy, Combination Therapy, Drug Resistance, Cancer Stem Cells, Aging, Senescence, Raf, Akt, PI3K, mTOR Received: February 25, 2011; Accepted: March 10, 2011; Published: March 11, 2011; Correspondence: James A. McCubrey, e-mail: // // Abstract The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Integral components of these pathways, Ras, B-Raf, PI3K, and PTEN are also activated/inactivated by mutations. These pathways have profound effects on proliferative, apoptotic and differentiation pathways. Dysregulation of these pathways can contribute to chemotherapeutic drug resistance, proliferation of cancer initiating cells (CICs) and premature aging. This review will evaluate more recently described potential uses of MEK, PI3K, Akt and mTOR inhibitors in the proliferation of malignant cells, suppression of CICs, cellular senescence and prevention of aging. Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways play key roles in the regulation of normal and malignant cell growth. Inhibitors targeting these pathways have many potential uses from suppression of cancer, proliferative diseases as well as aging.

Mesenchymal stem cells (MSCs) are adult multipotent cells that give rise to various cell types of the mesodermal germ layer. MSCs are of great interest in the field of regenerative medicine and cancer therapy because of their unique ability to home to damaged and cancerous tissue. These cells also regulate the immune response and contribute to reparative processes in different pathological conditions, including musculoskeletal and cardiovascular diseases. The use of MSCs for tissue repair was initially based on the hypothesis that these cells home to and differentiate within the injured tissue into specialized cells. However, it now appears that only a small proportion of transplanted MSCs actually integrate and survive in host tissues. Thus, the predominant mechanism by which MSCs participate in tissue repair seems to be related to their paracrine activity. Indeed, MSCs provide the microenvironment with a multitude of trophic and survival signals including growth factors and cytokines. Recent discoveries suggest that lipid microvesicles released by MSCs may also be important in the physiological function of these cells. Over the past few years the biological relevance of micro- and nano-vesicles released by cells in intercellular communication has been established. Alongside the conventional mediators of cell secretome, these sophisticated nanovesicles transfer proteins, lipids and, most importantly, various forms of RNAs to neighboring cells, thereby mediating a variety of biological responses. The physiological role of MSC-derived vesicles (MSC-MVs) is currently not well understood. Nevertheless, encouraging results indicate that MSC-MVs have similar protective and reparative properties as their cellular counterparts in tissue repair and possibly anti-cancer therapy. Thus, MSC-MVs represent a promising opportunity to develop novel cell-free therapy approaches that might overcome the obstacles and risks associated with the use of native or engineered stem cells.