Exosomes isolation and characterization in serum is feasible in non-small cell lung cancer patients: critical analysis of evidence and potential role in clinical practice

Exosomes are nano-sized vesicles of endolysosomal origin, released by several cytotypes in physiological and pathological conditions. Tumor derived exosomes, interacting with other cells of the tumor microenvironment, modulate tumor progression, angiogenic switch, metastasis, and immune escape. Recently, extracellular vesicles were proposed as excellent biomarkers for disease monitoring and prognosis in cancer patients. Non-small cell lung cancer (NSCLC) has a poor 5-year survival rate due to the delay in the detection of the disease. The majority of patients are diagnosed in an advanced disease stage. Exosomes might be promising beneficial tools as biomarker candidates in the scenario of NSCLC, since they contain both, proteins and miRNAs. The clinical case reported in this manuscript is a proof of concept revealing that NSCLC exosomes and sorted miRNAs might constitute, in a near future, novel biomarkers. This review summarizes the role of exosomes in NSCLC, focusing on the importance of exosomal microRNAs in lung cancer diagnosis and prognosi

Quantitative Proteomics Reveals Changes Induced by TIMP-3 on Cell Membrane Composition and Novel Metalloprotease Substrates

Ectodomain shedding is a key mechanism of several biological processes, including cell-communication. Disintegrin and metalloproteinases (ADAMs), together with the membrane-type matrix metalloproteinases, play a pivotal role in shedding transmembrane proteins. Aberrant shedding is associated to several pathological conditions, including arthritis. Tissue inhibitor of metalloproteases 3 (TIMP-3), an endogenous inhibitor of ADAMs and matrix metalloproteases (MMPs), has been proven to be beneficial in such diseases. Thus, strategies to increase TIMP-3 bioavailability in the tissue have been sought for development of therapeutics. Nevertheless, high levels of TIMP-3 may lead to mechanism-based side-effects, as its overall effects on cell behavior are still unknown. In this study, we used a high-resolution mass-spectrometry-based workflow to analyze alterations induced by sustained expression of TIMP-3 in the cell surfaceome. In agreement with its multifunctional properties, TIMP-3 induced changes on the protein composition of the cell surface. We found that TIMP-3 had differential effects on metalloproteinase substrates, with several that accumulated in TIMP-3-overexpressing cells. In addition, our study identified potentially novel ADAM substrates, including ADAM15, whose levels at the cell surface are regulated by the inhibitor. In conclusion, our study reveals that high levels of TIMP-3 induce modifications in the cell surfaceome and identifies molecular pathways that can be deregulated via TIMP-3-based therapies

A weekly regimen of cisplatin, paclitaxel and topotecan with granulocyte-colony stimulating factor support for patients with extensive disease small cell lung cancer: a phase II study

The present study was aimed at defining the antitumour activity of the cisplatin-paclitaxel-topotecan (CPT) weekly administration with G-CSF support in chemo-naive SCLC patients with extensive disease (ED-SCLC). Chemonaive ED-SCLC patients received cisplatin 40 mg/m2, paclitaxel 85 mg/m2, and topotecan 2.25 mg/m2weekly, with G-CSF (5 μg/kg days 3–5) support, for a maximum of 12 weeks. 37 patients were treated, for a total of 348 cycles delivered. 8 complete responses (22%) and 22 partial responses (59%) were recorded, giving an 81% [95% CI = 65–92%] ORR. At a 13-month (range, 4–26) median follow-up, median progression-free and overall survival were 8 months and 12.5 months, with 1-year and 2-year projected survivals of 55% and 21%, respectively. No toxic deaths occurred. Grade 4 neutropenia and thrombocytopenia occurred in 6 and 3 patients, respectively. Only one case of neutropenic sepsis was recorded, while haemorrhagic thrombocytopenia was never observed. Diarrhoea, paraesthesias and fatigue were the main nonhaematologic toxicities being severe in 6, 2 and 10 patients, respectively. The weekly CPT combination with G-CSF support represents a well tolerated therapeutic approach in chemo-naive ED-SCLC patients. The activity rate seems at least similar to that achievable with the standard front-line approaches. © 2001 Cancer Research Campaign http://www.bjcancer.co

Phenotypical and molecular assessment of the virulence potential of KPC-3-producing Klebsiella pneumoniae ST392 clinical isolates

Klebsiella pneumoniae is a Gram-negative bacterium of clinical importance, due to its resistance to several antibiotic classes. We have identified 4 clinical isolates of K. pneumoniae sequence type (ST) 392 KPC-3-producing strains from patients at the Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione (IRCCS-ISMETT), a Southern Italian transplantation health facility, during a routine surveillance for carbapenemase-producing Enterobacterales from in-house clinical samples. Since those were among, to the best of our knowledge, the first KPC-producing K. pneumoniae ST392 isolated in Europe, we assessed their virulence potential, to understand if this particular ST can become an endemic clinical threat. ST392 isolates were investigated to assess their virulence potential, namely resistance to human sera, formation of abiotic biofilms, adhesion to biotic surfaces, exopolysaccharide production and in vivo pathogenesis in the wax moth Galleria mellonella animal model. ST392-belonging strains were highly resistant to human sera. These strains also have a high capacity to form abiotic biofilms and high levels of adhesion to the human epithelial colorectal adenocarcinoma HT-29 cell line. An increase of transcriptional levels of genes involved in serum resistance (aroE and traT) and adhesion (pgaA) was observed when compared with the Klebsiella quasipneumoniae subsp. similipneumoniae strain ATCC 700603 reference strain. Infection of G. mellonella larvae with ST392 clinical isolates showed that the latter were not highly pathogenic in this model. Together, our results indicate that ST392 isolates have the potential to become a strain of clinical relevance, especially in health settings where patients are immunosuppressed, e.g., transplant recipients

Quantitative proteomics reveals that ADAM15 can have proteolytic-independent functions in the steady state

A disintegrin and metalloproteinase 15 (ADAM15) is a member of the ADAM family of sheddases. Its genetic ablation in mice suggests that ADAM15 plays an important role in a wide variety of biological functions, including cartilage homeostasis. Nevertheless, while the substrate repertoire of other members of the ADAM family, including ADAM10 and ADAM17, is largely established, little is known about the substrates of ADAM15 and how it exerts its biological functions. Herein, we used unbiased proteomics to identify ADAM15 substrates and proteins regulated by the proteinase in chondrocyte-like HTB94 cells. ADAM15 silencing did not induce major changes in the secretome composition of HTB94 cells, as revealed by two different proteomic approaches. Conversely, overexpression of ADAM15 remodeled the secretome, with levels of several secreted proteins being altered compared to GFP-overexpressing controls. However, the analysis did not identify potential substrates of the sheddase, i.e., transmembrane proteins released by ADAM15 in the extracellular milieu. Intriguingly, secretome analysis and immunoblotting demonstrated that ADAM15 overexpression increased secreted levels of tissue inhibitor of metalloproteinases 3 (TIMP-3), a major regulator of extracellular matrix turnover. An inactive form of ADAM15 led to a similar increase in the inhibitor, indicating that ADAM15 regulates TIMP-3 secretion by an unknown mechanism independent of its catalytic activity. In conclusion, high-resolution quantitative proteomics of HTB94 cells manipulated to have increased or decreased ADAM15 expression did not identify canonical substrates of the proteinase in the steady state, but it revealed that ADAM15 can modulate the secretome in a catalytically-independent manner

Participation of older newly-diagnosed cancer patients in an observational prospective pilot study: an example of recruitment and retention

<p>Abstract</p> <p>Background</p> <p>There have been few prospective observational studies which recruited older newly-diagnosed cancer patients, and of these only some have reported information on the number needed to screen to recruit their study sample, and the number and reasons for refusal and drop-out. This paper reports on strategies to recruit older newly-diagnosed cancer patients prior to treatment into an observational prospective pilot study and to retain them during a six-month period.</p> <p>Methods</p> <p>Medical charts of all patients in the Segal Cancer Centre aged 65 and over were screened and evaluated for inclusion. Several strategies to facilitate recruitment and retention were implemented. Reasons for exclusion, refusal and loss to follow-up were recorded. Descriptive statistics were used to report the reasons for refusal and loss to follow-up. A non-response analysis using chi-square tests and t-tests was conducted to compare respondents to those who refused to participate and to compare those who completed the study to those who were lost to follow-up. A feedback form with open-ended questions was administered following the last interview to obtain patient's opinions on the length of the interviews and conduct of this pilot study.</p> <p>Results</p> <p>3060 medical charts were screened and 156 eligible patients were identified. Of these 112 patients participated for a response rate of 72%. Reasons for refusal were: feeling too anxious (40%), not interested (25%), no time (12.5%), too sick (5%) or too healthy (5%) or other reasons (5%). Ninety-one patients participated in the six-month follow-up (retention 81.3%), seven patients refused follow-up (6.2%) and fourteen patients died (12.5%) during the course of the study. The median time to conduct the baseline interview was 45 minutes and 57% of baseline interviews were conducted at home. Most patients enjoyed participation and only five felt that the interviews were too long.</p> <p>Conclusion</p> <p>It was feasible to recruit newly-diagnosed cancer patients prior to treatment although it required considerable time and effort. Once patients were included, the retention rate was high despite the fact that most were undergoing active cancer treatment.</p

Combined treatment with fulvestrant plus capecitabine in elderly advanced breast cancer (EABC): Three-year evaluation of efficacy and safety

Background: EABC affects about 45% of all advanced breast cancers and actually treatments are always more toxic than effectiveness. Fulvestrant (F), the first of a new class of pure estrogen receptor antagonists, competitively binds to estrogen receptors on tumors and inhibit tumor growth. Capecitabine (C) is converted to 5-FU by thymidine phosphorylase and shows good efficacy and low toxicity as single agent in EABC. Since elderly cancer pts are extremely susceptible in development of rapid toxicity during treatment with cytotoxic drugs, we design this chemo-hormone combined schedule to evaluate if F + C could increase efficacy profile in EABC women. Methods: 41 EABC pts (mean age 72; range 65-88) PD after TAM or SERM were included in the study starting in 2007. Written informed consent for treatment's schedule was acquired from all pts. Comprehensive Geriatric Assessment (CGA) was performed to assess eligibility for chemotherapy. Furthermore the C dose was adjusted according to Cocroft and Kintzel-Dorr formulas. Primary endpoint: clinical response rates (RR) according to WHO criteria. Secondary endpoints: TTP, Toxicity Profile and QoL score measured by NCI-CTC v2.0 and EORTC QLQ-C30 questionnaires. Results: 41/41 pts were evaluated. All pts received C 1,000 mg/m2 b.i.d p.o. on days 1st to 14th every 21 days and F 250 mg i.m. once-monthly until disease progression. Based on CGA all patients were included into groups I-II, according to Balducci's classification of frailty. None of the pts needed any delay in drugs delivery. We observed 13/41 (32%) partial responses and 17/41 (41%) stable disease, for a Clinical benefit of 73%. Median time to progression was 15,6 months. No grade-4 toxicity was experienced. QoL score improvement was appreciated in all pts after end of treatment. Conclusions: Efficacy and Safety results show that this schedule is feasible for EABC pts affected by one or two comorbidities and also in frail condition. All pts are still alive and under monitoring for long-term toxicity and QoL. These results shows that F+C schedule increase clinical benefit-enhancing effectiveness and safety. Although a larger number of EABC pts must be enrolled to consolidate these findings