Silvia Cervo

Surface-enhanced Raman spectroscopy of blood plasma and serum using Ag and Au nanoparticles: a systematic study

Surface-enhanced Raman spectroscopy (SERS) is a good candidate for the development of fast and easy-to-use diagnostic tools, possibly used on biofluids in point-of-care or screening tests. In particular, label-free SERS spectra of blood serum and plasma, two biofluids widely used in diagnostics, could be used as a metabolic fingerprinting approach for biomarker discovery. This study aims at a systematic evaluation of SERS spectra of blood serum and plasma, using various Ag and Au aqueous colloids, as SERS substrates, in combination with three excitation lasers of different wavelengths, ranging from the visible to the near-infrared. The analysis of the SERS spectra collected from 20 healthy subjects under a variety of experimental conditions revealed that intense and repeatable spectra are quickly obtained only if proteins are filtered out from samples, and an excitation in the near-infrared is used in combination with Ag colloids. Moreover, common plasma anticoagulants such as EDTA and citrate are found to interfere with SERS spectra; accordingly, filtered serum or heparin plasma are the samples of choice, having identical SERS spectra. Most bands observed in SERS spectra of these biofluids are assigned to uric acid, a metabolite whose blood concentration depends on factors such as sex, age, therapeutic treatments, and various pathological conditions, suggesting that, even when the right experimental conditions are chosen, great care must be taken in designing studies with the purpose of developing diagnostic tests.

Label-free surface-enhanced Raman spectroscopy of biofluids: fundamental aspects and diagnostic applications

In clinical practice, one objective is to obtain diagnostic information while minimizing the invasiveness of the tests and the pain for the patients. To this end, tests based on the interaction of light with readily available biofluids including blood, urine, or saliva are highly desirable. In this review we examine the state of the art regarding the use of surface-enhanced Raman spectroscopy (SERS) to investigate biofluids, focusing on diagnostic applications. First, a critical evaluation of the experimental aspects involved in the collection of SERS spectra is presented; different substrate types are introduced, with a clear distinction between colloidal and non-colloidal metal nanostructures. Then the effect of the excitation wavelength is discussed, along with anomalous bands and artifacts which might affect SERS spectra of biofluids. The central part of the review examines the literature available on the SERS spectra of blood, plasma, serum, urine, saliva, tears, and semen. Finally, diagnostic applications are critically discussed in the context of the published evidence; this section clearly reveals that SERS of biofluids is most promising as a rapid, cheap, and non-invasive tool for mass screening for cancer.

HE4, CA125 and risk of ovarian malignancy algorithm (ROMA) as diagnostic tools for ovarian cancer in patients with a pelvic mass: An Italian multicenter study

OBJECTIVE This multicenter study aims to evaluate HE4, CA125 and risk of ovarian malignancy algorithm (ROMA) performance in the differential diagnosis of epithelial ovarian cancer (EOC). METHODS A total of 405 patients referred to gynecological oncologist with suspicious pelvic mass requiring a surgery for identification of EOC were consecutively enrolled; 387 patients satisfied inclusion criteria: 290 benign diseases; 15 borderline neoplasia and 82 tumors (73 EOC). RESULTS Good diagnostic performance in discriminating benign from EOC patients was obtained for CA125, HE4 and ROMA when calculating optimal cut-off values: premenopause, specificity (SP) >86.6, sensitivity (SN) >82.6, area under the curves (AUC)≥0.894; postmenopause, SP>93.2, SN>82, AUC≥0.928. Fixing SP at 98%, performance indicators obtained for benign vs EOC patients were: premenopause, SN:65.2%, positive predictive value (+PV): 75%, positive likelihood ratio (+LR): 26.4 for CA125; SN:69.6%, +PV:76.2%, +LR:28.1 for HE4; SN:69.6%, +PV: 80%; +LR:35.1 for ROMA; postmenopause, SN:88%, +PV: 95.7%, +LR:38.7 for CA125; SN:78%, +PV:95.1%, +LR:34.3 for HE4; SN:88%, +PV:97.8%, +LR:77.4 for ROMA. When using routine cut-off thresholds, ROMA showed better well-balanced values of both SP and SN (premenopause, SN:87%, SP:86.1%; postmenopause, SN:90%; SP:94.3%). CONCLUSIONS Overall, ROMA showed well balanced diagnostic performance to differentiate EOC from benign diseases. Meaningful differences of +PVs and +LRs between HE4 and CA125 suggest that the two markers may play at least in part different roles in EOC diagnosis, with HE4 seeming to be more efficient than CA125 in ruling in EOC patients in the disease group, also in early stages tumors, both in pre and postmenopause.

An effective multisource informed consent procedure for research and clinical practice: an observational study of patient understanding and awareness of their roles as research stakeholders in a cancer biobank

BackgroundEfforts to improve patients’ understanding of their own medical treatments or research in which they are involved are progressing, especially with regard to informed consent procedures. We aimed to design a multisource informed consent procedure that is easily adaptable to both clinical and research applications, and to evaluate its effectiveness in terms of understanding and awareness, even in less educated patients.MethodsWe designed a multisource informed consent procedure for patients’ enrolment in a Cancer Institute Biobank (CRO-Biobank). From October 2009 to July 2011, a total of 550 cancer patients admitted to the Centro di Riferimento Oncologico IRCCS Aviano, who agreed to contribute to its biobank, were consecutively enrolled. Participants were asked to answer a self-administered questionnaire aim at exploring their understanding of biobanks and their needs for information on this topic, before and after study participation. Chi-square tests were performed on the questionnaire answers, according to gender or education.ResultsOf the 430 patients who returned the questionnaire, only 36.5% knew what a biobank was before participating in the study. Patients with less formal education were less informed by some sources (the Internet, newspapers, magazines, and our Institute). The final assessment test, taken after the multisource informed consent procedure, showed more than 95% correct answers. The information received was judged to be very or fairly understandable in almost all cases. More than 95% of patients were aware of participating in a biobank project, and gave helping cancer research (67.5%), moral obligation, and supporting cancer care as main reasons for their involvement.ConclusionsOur multisource informed consent information system allowed a high rate of understanding and awareness of study participation, even among less-educated participants, and could be an effective and easy-to-apply model for others to consider to contribute to a well-informed decision making process in several fields, from clinical practice to research.Further studies are needed to explore the effects on the study comprehension by each source of information, and by other sources suggested by participants in the questionnaire.

Blocking tumor-educated MSC paracrine activity halts osteosarcoma progression

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets. Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)–educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography. Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients. Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721–33. ©2017 AACR.

Pepsinogens to Distinguish Patients With Gastric Intestinal Metaplasia and Helicobacter pylori Infection Among Populations at Risk for Gastric Cancer

Objectives:The objectives of this study were to investigate the serum pepsinogen test for the prediction of OLGIM (Operative Link on Gastric Intestinal Metaplasia Assessment) stages in first-degree relatives (FDR-GC) of patients with gastric cancer (GC) and autoimmune chronic atrophic gastritis (ACAG).Methods:In 67 consecutive patients with ACAG, 82 FDR-GC, and 53 controls (CTRL) without gastric disease (confirmed by biopsy), serum levels of pepsinogen 1 (PG1), pepsinogen 2 (PG2), G17, and the PG1/2 ratio were assessed by enzyme-linked immunosorbent assay kit. All ACAG patients had positive antiparietal cell antibody levels, estimated by indirect immunofluorescence. Biopsies taken in duplicate from the antrum, corpus, and fundus were stained with Giemsa for Helicobacter pylori detection. Endoscopic detection of metaplasia was confirmed by histological diagnosis. Histological classification of OLGIM stages was applied by using the criteria of severity and topography of intestinal metaplasia (IM).Results:The highest discrimination capacity for distinguishing ACAG from other groups of patients was the gastrin G17 test. The lowest mean for PG1 and PG2 serum levels was found in ACAG. In multivariate analysis by age, PG1 and PG1/PG2 were independent prognostic factors for metaplasia, and PG2 also for the presence of a histological H. pylori infection. The serum PG1 level was significantly lower in individuals with IM at OLGIM stage >2 than in those with IM at OLGIM stage <2, resulting in a useful method for the prediction of OLGIM stage. With the inclusion of patient age at diagnosis in the prediction of ≥2 vs. 0–1 OLGIM stages, the receiver operating characteristic (ROC) curve at 47.9 ng/ml PG1 level reached a significant area under the curve (AUC) value (0.978, P<0.001). We also observed a slight difference in PG2 serum levels between histological H. pylori-positive and H. pylori-negative subjects (ROC AUC: 0.599).Conclusions:This study demonstrated an important increase in gastrin G17 serum level in autoimmune gastritis. PG1 serum level corrected by patient age can be used in the management of patients at risk for GC with a high predicted probability of having an OLGIM stage ≥2. Using a cutoff of 47.9 ng/ml, PG1 testing in FDR-GC and ACAG patients had a sensitivity of 95.83% and a specificity of 93.37. Although these results could be validated in a prospective study, the known importance of higher OLGIM stages in increasing the risk of GC development supports the rationale of proposing PG1 algorithm as a diagnostic tool for the selection of high-risk FDR-GC and ACAG patients at high-risk stages for subsequent detailed endoscopic examination to detect dysplasia and asymptomatic GC. In addition, serum PG1 and PG2 levels could stratify patients based on both H. pylori infection and OLGIM risk in consideration of the increased acknowledge regarding the role of H. pylori in the progression of gastritis to GC.

Patient-Centered Cancer Care Programs in Italy: Benchmarking Global Patient Education Initiatives.

In Italy, educational programs for cancer patients are currently provided by the national government, scientific societies, and patient advocate organizations. Several gaps limit their effectiveness, including the lack of coordinated efforts, poor involvement of patient feedback in the planning of programs, as well as a lack of resources on innovative cancer-related topics. This process is parallel to a strong shift in the attitude of patients towards health in general and taking charge of their own health conditions in particular. The National Cancer Institute in the USA and the Organization of European Cancer Institutes encourage comprehensive cancer centers in providing educational programs conceived to overcome these gaps. The goal of this paper is to identify and describe the key elements necessary to develop a global patient education program and provide recommendations for strategies with practical examples for implementation in the daily activities of cancer institutes. A multidisciplinary committee was established for patient education, including patient representatives as equal partners, to define, implement, verify, and evaluate the fundamental steps for establishing a comprehensive education program. Six essential topics were identified for the program: appropriate communication of cancer epidemiology, clinical trial information, new therapeutic technologies, support in the use of medicines, psycho-oncological interventions, age-personalized approaches, and training programs for healthcare providers. Integration of these topics along with patient feedback is the key to a successful model for educational programs. An integrated educational program can transform a comprehensive cancer center to an institution that provides research and care for and with patients.

Cost-effective organization of an institutional human cancer biobank in a clinical setting: CRO-Biobank experience toward harmonization

This report describes the organization of the Biobank of the CRO Aviano National Cancer Institute, Aviano (CRO- Biobank), Italy, implemented as a structured facility dedicated to collecting human biological samples. It describes a particular disease-specific biobank and the integration of a research biobank in a clinical setting. The CRO-Biobanks mission is rooted in supporting and implementing cancer research, with its main focus on optimizing technical and quality processes, while also investigating ethical, legal and IT topics. The CRO-Biobank has implemented processes aimed at guaranteeing the safety of the providers, protecting patient privacy and ensuring both the traceability and quality of its samples. Our 8 years of experience allow us to offer insights and useful suggestions that may solve theoretical and practical issues that can arise when starting up new biobanks or developing existing biobanks further.

Conservative treatment of coexisting microinvasive squamous and adenocarcinoma of the cervix: report of two cases and literature review

Coexistence of microinvasive squamous cell carcinoma (MISCC) and microinvasive adenocarcinoma (MIAC) of the cervix is a rare phenomenon with very few clinically significant cases described in the literature. While a conservative approach has been studied, and may be effective in MISCC, a lower number of studies that recommend conservative treatment are available for MIAC. We report two cases of synchronous cervix lesions in two separate foci, MISCC and MIAC, who underwent fertility-sparing treatment with long-term follow-up. We describe clinical, histological, and immunohistochemical features of the two cases. The first case is a 41-year-old female with a diagnosis of MIAC of endocervical type, grade 1 differentiation, with a stromal invasion, associated with a separate area of squamous cell carcinoma (International Federation of Gynecology and Obstetrics/TNM stage: pT1a1G1). The second case is a 45-year-old female with a diagnosis of plurifocal MISCC, associated with an MIAC of endocervical type with a stromal invasion (International Federation of Gynecology and Obstetrics/TNM stage: pT1a1G1). After multidisciplinary counseling, both patients accepted conization as definitive treatment. Eleven years after the conization, all tests (Papanicolaou smear, colposcopy, cervical curettage, and hybrid capture 2-human papillomavirus test) planned quarterly in the first year and every 6 months in the subsequent years were negative in both patients. In women affected by stage IA1 squamous cervical cancer coexisting with stage IA1 adenocarcinoma endocervical type, with clear margins, and without lymphovascular space invasion, cervical conization may be considered a fertility-preserving, safe, and definitive therapeutic option.

Drafting biological material transfer agreement: a ready-to-sign model for biobanks and biorepositories

Purpose Due to the scarcity of publications, guidelines, and harmonization among national regulations, biobanks and institutions face practical and theoretical issues when drafting a material transfer agreement (MTA), the fundamental tool to regulate the successful exchange of biosamples and information. Frequently researchers do not execute MTAs because of a general lack of knowledge about this topic. It is thus critical to develop new models to prevent loss of traceability and opportunities both for researchers and biobanks, their exposure to various risks, and delays in transferring biomaterials. Methods Through the involvement of institutional groups and professionals with multidisciplinary expertise, we have drawn up a ready-to-sign MTA for the CRO-Biobank (the biobank of the National Cancer Institute, CRO, Aviano), a standardized template that can be employed as a ready-to-use model agreement. Results The team identified the essential components to be included in the MTA, which comprise i) permissions, liability and representations; ii) custodianship and distribution limitations; iii) appropriate use of materials, including biosafety concerns; iv) confidentiality, non-disclosure, and publications; v) intellectual property protection for both the provider and recipient. Conclusions This paper aims to be an unabridged report (among the few works in the existing literature) providing a description of the whole process related to the formation of an MTA. Biobanks and institutions may consider adopting our ready-to-sign form as a standard model. The article discusses the most important issues tackled during the drafting of the document, thus proposing an operative approach for other institutions that face the same problems.