Publications by authors named "Elisabetta Rossi"

100 Publications

A fully automated assay to detect the expression of pan-cytokeratins and of EML4-ALK fusion protein in circulating tumour cells (CTCs) predicts outcome of non-small cell lung cancer (NSCLC) patients.

Transl Lung Cancer Res 2021 Jan;10(1):80-92

Veneto Institute of Oncology IOV - IRCCS, Padua, Italy.

Background: In advanced non-small cell lung cancer (NSCLC) a recent meta-analysis confirms circulating tumour cells (CTCs) as an independent prognostic indicator of progression-free survival (PFS) and overall survival (OS). However, further investigations are necessary to predict and dynamically monitor the therapy in NSCLC patients using CTCs. To this aim, we combined the classical standard assay (SA) with an expanded cytokeratins profile (EA) and quantified the expression of EML4-ALK fusion protein in CTCs.

Methods: The CellSearch (CS) platform-first marked diagnostic use (IVD) from Food and Drug Administration (FDA), and "gold standard" for quantifying CTCs - detects EpCAM and cytokeratins (CKs) 8, 18, and 19. Since NSCLC shows different CKs profile, we customized the SA, to recognize CK 4, 5, 6, 7, 8, 10, 13, 14, 18, and 19 (EA). Using both assays we designed a prospective, multi-center study, primarily aimed to enumerate CTCs in advanced NSCLC. Secondarily, we developed an integration of the EA to quantify the expression of EML4-ALK fusion protein in CTCs, and correlated them with PFS and OS.

Results: EA identified a significantly much more number of CTC-positive patients (115 out of 180) than SA (103 out of 192; Chi-square =4.0179, with 1 degrees of freedom, P=0.04502). Similar to SA, EA levels were still associated with patient' outcomes. Furthermore, the expression of EML4-ALK on CTCs allowed stratifying NSCLC patients according to a statistically significant difference in PFS.

Conclusions: We proposed here two novel automated tests, to characterize the expression of specific molecules on CTCs. We demonstrated that these integrated assays are robust and actionable in prospective clinical studies, and in the future could allow clinicians to improve both choice and length of treatment in individual NSCLC patient.
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http://dx.doi.org/10.21037/tlcr-20-855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867748PMC
January 2021

Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer (Coleoptera: Cerambycidae) from frass.

3 Biotech 2021 Feb 19;11(2):85. Epub 2021 Jan 19.

Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy.

The red-necked longhorn beetle (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus . Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 10 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of and in the management of its outbreaks.
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http://dx.doi.org/10.1007/s13205-020-02602-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815857PMC
February 2021

Proficiency Testing to Assess Technical Performance for CTC-Processing and Detection Methods in CANCER-ID.

Clin Chem 2021 Mar;67(4):631-641

Department of General, Visceral and Paediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.

Background: Multiple technologies are available for detection of circulating tumor cells (CTCs), but standards to evaluate their technical performance are still lacking. This limits the applicability of CTC analysis in clinic routine. Therefore, in the context of the CANCER-ID consortium, we established a platform to assess technical validity of CTC detection methods in a European multi-center setting using non-small cell lung cancer (NSCLC) as a model.

Methods: We characterized multiple NSCLC cell lines to define cellular models distinct in their phenotype and molecular characteristics. Standardized tumor-cell-bearing blood samples were prepared at a central laboratory and sent to multiple European laboratories for processing according to standard operating procedures. The data were submitted via an online tool and centrally evaluated. Five CTC-enrichment technologies were tested.

Results: We could identify 2 cytokeratin expressing cell lines with distinct levels of EpCAM expression: NCI-H441 (EpCAMhigh, CKpos) and NCI-H1563 (EpCAMlow, CKpos). Both spiked tumor cell lines were detected by all technologies except for the CellSearch system that failed to enrich EpCAMlow NCI-H1563 cells. Mean recovery rates ranged between 49% and 75% for NCI-H411 and 32% and 76% for NCI-H1563 and significant differences were observed between the tested methods.

Conclusions: This multi-national proficiency testing of CTC-enrichment technologies has importance in the establishment of guidelines for clinically applicable (pre)analytical workflows and the definition of minimal performance qualification requirements prior to clinical validation of technologies. It will remain in operation beyond the funding period of CANCER-ID in the context of the European Liquid Biopsy Society (ELBS).
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http://dx.doi.org/10.1093/clinchem/hvaa322DOI Listing
March 2021

Baseline CD44v6-positive circulating tumor cells to predict first-line treatment failure in patients with metastatic colorectal cancer.

Oncotarget 2020 Nov 10;11(45):4115-4122. Epub 2020 Nov 10.

Department of Molecular Medicine, Liquid Biopsy Unit, Sapienza University of Rome, Rome, Italy.

CD44v6, the CD44 isoform mostly involved in cancer cell migration and invasion, has been identified as a functional biomarker and therapeutic target in colon cancer stem cells. We here provide evidence that baseline CD44v6-positive CTC predict treatment failure in patients with metastatic colorectal cancer undergoing first-line chemotherapy. We suggest that CD44v6-positive CTC can be used to early detect intrinsic drug resistance in this cancer type.
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http://dx.doi.org/10.18632/oncotarget.27794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665234PMC
November 2020

A Novel Method of Implant Coverage for Post-Mastectomy Reconstruction after Previous Augmentation: A Case Report.

Breast Care (Basel) 2020 Oct 28;15(5):534-537. Epub 2020 Jan 28.

Division of Breast Cancer Reconstruction, European Institute of Oncology, IRCCS, Milan, Italy.

Background: Breast augmentation is the most common -cosmetic surgical procedure in the USA, with nearly 300,000 women undergoing surgery annually. National incidence rates predict that among women undergoing breast augmentation each year, approximately 35,000 will eventually be diagnosed with breast cancer, in particular individual / germline mutant carriers.

Case Report: Our case introduces a novel method of implant coverage after immediate post-mastectomy reconstruction in augmented patients. A novel "capsular flap" (flap of the pre-existing old capsule) is isolated and refolded to cover the outer lower portion of the implant.

Conclusion: Tailored surgical approaches can be offered to those patients previously augmented and requiring mastectomy after breast cancer diagnosis.
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http://dx.doi.org/10.1159/000505226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650099PMC
October 2020

Liquid Biopsy in Pediatric Renal Cancer: Stage I and Stage IV Cases Compared.

Diagnostics (Basel) 2020 Oct 12;10(10). Epub 2020 Oct 12.

Veneto Institute of Oncology IOV-IRCCS, Padua, Italy.

Pediatric renal cancer is rare, and robust evidence for treatment recommendations is lacking. In the perspective of personalized medicine, clinicians need new biomarkers to improve risk stratification and patients' follow-up. Herein, we analyzed some liquid biopsy tools, which have been never tested in pediatric renal cancer: namely, circulating tumor cells (CTCs); the expression of M30, an apoptosis marker, to test CTC metastatic potential; and c-MET expression in CTCs, because of its role in renal cancer progression and drug-resistance. Furthermore, we evaluated the Circulating Endothelial Cells (CECs), whose utility we previously demonstrated in adult metastatic renal cancer treated with anti-angiogenic therapy. We compared two renal cell carcinomas of clear-cell type, stage I and IV, which underwent surgery and surgery plus Sunitinib, respectively. Baseline CTC level and its changes during follow-up were consistent with patients' outcome. In case 2, stage IV, the analysis of CECs performed during Sunitinib revealed a late response to treatment consistent with poor outcome, as the finding of M30-negative, viable cells. Noteworthily, few CTCs were MET-positive in both cases. Our study highlights the feasibility for a change in the prognostic approach and follow-up of childhood renal cancer, with a view to guide a better treatment design.
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http://dx.doi.org/10.3390/diagnostics10100810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599903PMC
October 2020

Molecular Identification of Anoplophora glabripennis (Coleoptera: Cerambycidae) From Frass by Loop-Mediated Isothermal Amplification.

J Econ Entomol 2020 12;113(6):2911-2919

Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto, Pisa, Italy.

Anoplophora glabripennis (Motschulsky, 1853), native to eastern Asia, is a destructive woodborer of many ornamental species, leading to the decline and the death of the attacked trees. In outbreak areas as Europe or North America, this pest is usually identified using morphological or molecular analyses of adult or larval specimens. However, the procedures for collecting A. glabripennis specimens from infested plants are too expensive and time consuming for routine screening. A noninvasive diagnostic tool based on frass discrimination is therefore crucial for the rapid identification of A. glabripennis at different development stages in the host. This article describes a rapid diagnostic protocol based on loop-mediated isothermal amplification (LAMP). DNA extracted from A. glabripennis frass was amplified with both visual and real-time LAMP and compared with those of nontarget species. The results show that the method is reliable and accurate and therefore could be a promising diagnostic tool in phytosanitary surveys.
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http://dx.doi.org/10.1093/jee/toaa206DOI Listing
December 2020

Pediatric sarcomas display a variable EpCAM expression in a histology-dependent manner.

Transl Oncol 2020 Nov 14;13(11):100846. Epub 2020 Aug 14.

Department of Woman's and Children's Health, Hematology and Oncology Unit, University of Padua, Padua, Italy.

EpCAM is a transmembrane glycoprotein typically overexpressed in cancer of epithelial origin and mainly involved in the epithelial-to-mesenchymal transition (EMT) of tumor cells that spread and disseminate. Strategies for the targeting and capture of EpCAM-expressing tumor cells are showing promise in cancers prone to metastatize, both as diagnostic tools and potential therapies. Sarcomas are among the most aggressive tumors in children, with a common mesenchymal origin that comprises both soft tissue sarcomas (STS) and bone sarcomas. The aim of this study was to assess EpCAM expression in pediatric sarcomas and correlate its expression with disease progression. To do so, we analyzed a set of cell lines and primary tumor tissues from rhabdomyosarcoma (RMS), Ewing sarcoma (ES), synovial sarcoma (SS) and desmoplastic small round cell tumor (DSRCT) STS, or osteosarcoma (OS) bone cancer. We demonstrated that EpCAM was variably expressed in pediatric sarcomas, with DSRCT, a rare, aggressive and almost fatal tumor type, characterized by the highest EpCAM expression levels. Interestingly, although EpCAM expression was lower in RMS tumors, high levels at diagnosis correlated with reduced patients' overall survival (p < 0.05). Indeed, membrane-bound EpCAM was detected in circulating sarcoma tumor cells, revealing its potential to be used as dissemination biomarker in this type of childhood cancers. This reinforces the concept that pediatric sarcomas do express both epithelial and mesenchymal markers and reside in an intermediate condition that most likely contributes to their aggressive phenotype and low survival rate.
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http://dx.doi.org/10.1016/j.tranon.2020.100846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453064PMC
November 2020

Possible role of circulating tumor cells in early detection of lung cancer.

J Thorac Dis 2020 Jul;12(7):3821-3835

Veneto Institute of Oncology IOV-IRCCS, Padua, Italy.

The prognosis of lung cancer varies highly depending on the disease stage at diagnosis, from a 5-year survival rate close to 90% in stage I, to 10% or less in stage IV disease. The enhancement of early diagnosis of this malignancy is mandatory to improve prognosis, because lung cancer patients stay long asymptomatic or few symptomatic after disease onset. Nowadays, liquid biopsy has emerged as a minimally-invasive tool to address the urgent need for real time monitoring, stratification, and personalized treatment of malignancies, including lung cancer. Liquid biopsy refers to a class of biomarkers, including circulating tumor cells (CTCs), cell-free circulating tumor DNA (ctDNA) and tumor-derived extracellular vesicles (tdEV). Since CTCs represent a crucial step in disease progression and metastasis, we reviewed here the scientific literature about the use of CTCs in early diagnosis of lung cancer; different techniques, and different strategies (e.g., source of analysis sample or high-risk groups of patients) were discussed showing the potential of implementing liquid biopsy in the clinical routine of non-metastatic lung cancer.
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http://dx.doi.org/10.21037/jtd.2020.02.24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399415PMC
July 2020

Potential treatment strategy for the rare osimertinib resistant mutation EGFR L718Q.

J Thorac Dis 2020 May;12(5):2771-2780

Department of Thoracic Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China.

Epidermal growth factor receptor (EGFR) L718Q is a rare resistant mutation which independently leads to third-generation tyrosine kinase inhibitor (TKI) resistance. Although a few studies have examined its resistance mechanisms, no effective treatment strategy has yet been proposed for patients with this mutation. Here, we report an effective treatment strategy for the rare EGFR L718Q mutation for the first time. A 44-year-old Chinese male patient initially presented with the sensitizing EGFR L858R mutation, and the progression-free survival (PFS) time after initial icotinib treatment was 9 months. When the progression of the disease (PD) and the EGFR T790M mutation were identified, he did not respond to the osimertinib treatment. Through comprehensive next-generation sequencing (NGS) of the surgical specimen, the rare EGFR L718Q mutation was eventually identified as having a frequency of 68.84%, together with an EGFR amplification with a copy number of 11.54. The previous treatment response was retrospectively explained, and the patient faced the challenge of not being able to benefit from any targeted therapy. Following chemotherapy with a personalized regimen which effectively modified the proportion of sensitive and resistant cells, significant response to osimertinib re-challenge was observed, and another PFS of 4.7 months was achieved. Unfortunately, four EGFR mutations, EGFR L858, T790M, L718Q, and C797S, were simultaneously detected in his late stage, and led to further progression of disease.
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http://dx.doi.org/10.21037/jtd.2020.03.29DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330377PMC
May 2020

How Useful Are Tumor Markers in Detecting Metastases with FDG-PET/CT during Breast Cancer Surveillance?

Oncology 2020 9;98(10):714-718. Epub 2020 Jun 9.

Division of Breast Surgery, European Institute of Oncology, IRCCS, Milan, Italy.

Background: To assess the clinical usefulness of serum tumor markers for early detection of distant breast cancer recurrence using FDG-PET/CT.

Methods: We retrospectively analyzed 561 consecutive patients who underwent surgery for invasive primary breast cancer and had increased tumor markers (CA 15-3 and CEA) after completion of locoregional therapy. FDG-PET/CT data were reviewed for all cases. CA 15-3 and CEA were evaluated both in a continuous and in a quartile (Q) distribution. The Wilcoxon rank-sum test and logistic regression models were used to evaluate the association between increased tumor marker values and the presence (and type) of distant metastases.

Results: The median value of CA 15-3 was 35.0 U/mL (IQR, 29.5-43.0) in cases where no distant metastases were detected, and it was 58.9 U/mL (IQR, 40.0-108.0) in cases where metastases were detected (p < 0.001). The median value of CEA was 6.6 U/mL (IQR, 4.4-10.0) in cases of no metastases and 12.4 U/mL (IQR, 6.9-30.0) in cases of metastases (p < 0.001). Increased levels of both tumor markers (Q3 and Q4) were strongly associated with the presence of distant metastases. The association between CA 15-3 and bone/liver metastases was stronger compared with other types of metastases (p heterogeneity between odds ratios [ORs] = 0.03 for Q3 and <0.001 for Q4), while no relevant heterogeneity between ORs emerged for CEA.

Conclusion: Increased tumor marker levels detected in asymptomatic breast cancer patients during adjuvant therapies and follow-up are significantly predictive of distant metastases identified on FDG-PET/CT.
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http://dx.doi.org/10.1159/000507707DOI Listing
October 2020

Dysmetabolic Circulating Tumor Cells Are Prognostic in Metastatic Breast Cancer.

Cancers (Basel) 2020 Apr 19;12(4). Epub 2020 Apr 19.

Immunopathology and Cancer Biomarkers, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, 33081 Aviano, Italy.

Circulating tumor cells (CTCs) belong to a heterogeneous pool of rare cells, and a unequivocal phenotypic definition of CTC is lacking. Here, we present a definition of metabolically-altered CTC (MBA-CTCs) as CD45-negative cells with an increased extracellular acidification rate, detected with a single-cell droplet microfluidic technique. We tested the prognostic value of MBA-CTCs in 31 metastatic breast cancer patients before starting a new systemic therapy (T0) and 3-4 weeks after (T1), comparing results with a parallel FDA-approved CellSearch (CS) approach. An increased level of MBA-CTCs was associated with: i) a shorter median PFS pre-therapy (123 days vs. 306; < 0.0001) and during therapy (139 vs. 266 days; = 0.0009); ii) a worse OS pre-therapy ( = 0.0003, 82% survival vs. 20%) and during therapy ( = 0.0301, 67% survival vs. 38%); iii) good agreement with therapy response (kappa = 0.685). The trend of MBA-CTCs over time (combining data at T0 and T1) added information with respect to separate evaluation of T0 and T1. The combined results of the two assays (MBA and CS) increased stratification accuracy, while correlation between MBA and CS was not significant, suggesting that the two assays are detecting different CTC subsets. In conclusion, this study suggests that MBA allows detection of both EpCAM-negative and EpCAM-positive, viable and label-free CTCs, which provide clinical information apparently equivalent and complementary to CS. A further validation of proposed method and cut-offs is needed in a larger, separate study.
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http://dx.doi.org/10.3390/cancers12041005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226515PMC
April 2020

Insulin-like growth factor-1 receptor (IGF-1R) expression on circulating tumor cells (CTCs) and metastatic breast cancer outcome: results from the TransMYME trial.

Breast Cancer Res Treat 2020 May 21;181(1):61-68. Epub 2020 Mar 21.

Unit of Biostatistics and Clinical Trials, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy.

Purpose: To evaluate the prognostic value of IGF-1R expression on circulating tumor cells (CTCs) in a prospective randomized clinical trial comparing chemotherapy plus metformin with chemotherapy alone in metastatic breast cancer (MBC) patients.

Methods: CTCs were collected at baseline and at the end of chemotherapy. An automated sample preparation and analysis system (CellSearch) were customized for detecting IGF-1R expression. The prognostic role of CTC count and IGF-1R was assessed for PFS and OS by univariate and multivariate analyses.

Results: Seventy-two out of 126 randomized patients were evaluated: 57% had ≥ 1 IGF-1R positive CTC and 37.5% ≥ 4 IGF-1R negative cells; 42% had CTC count ≥ 5/7.5 ml. At univariate analysis, the number of IGF-1R negative CTCs was strongly associated with risk of progression and death: HR 1.93 (P = 0.013) and 3.65 (P = 0.001), respectively; no association was detected between number of IGF-1R positive CTCs and PFS or OS (P = 0.322 and P = 0.840). The prognostic role of CTC count was confirmed: HR 1.69, P = 0.042 for PFS and HR 2.80 for OS, P = 0.002. By multivariate analysis, the prognostic role of the number of IGF-1R negative CTCs was maintained, while no residual prognostic role of CTC count or number of IGF-1R positive cells was found.

Conclusion: Loss of IGF-1R in CTCs is associated with a significantly worse outcome in MBC patients. This finding supports further evaluation for the role of IGF-1R on CTCs to improve patient stratification and to implement new targeted strategies.

Clinical Trial Registration: Clinicaltrials.gov (NCT01885013); European Clinical Trials Database (EudraCT No.2009-014,662-26).
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http://dx.doi.org/10.1007/s10549-020-05596-4DOI Listing
May 2020

Dynamic changes of Receptor activator of nuclear factor-κB expression in Circulating Tumor Cells during Denosumab predict treatment effectiveness in Metastatic Breast Cancer.

Sci Rep 2020 Jan 28;10(1):1288. Epub 2020 Jan 28.

Medical Oncology Department of Campus Bio-Medico, University of Rome, Rome, Italy.

Receptor-activator of nuclear-factor -κB-ligand (RANKL) and its receptor RANK have been recently identified as key players in breast cancer bone metastases. Since Circulating Tumor Cells (CTCs) are considered a crucial step of metastatic process, we explored RANK expression on CTCs in metastatic breast cancer (MBC), and the predictive value of RANK-positive CTCs in monitoring patients during treatment with denosumab (anti-RANKL antibody). To this purpose, we developed a novel CTC assay to quantify RANK-positive CTCs in forty-two bone MBC patients, candidates to denosumab treatment. Companion algorithms ΔAUC and Slope were developed, and correlated with time to first skeletal-related-events (SRE), time to bone metastasis progression and time to visceral metastasis progression. Twenty-seven patients had at least one CTC at baseline and, among these, nineteen (70%) had one or more RANK-positive CTCs. Notably, the baseline total CTCs, but not the RANK-positive, were associated with Time-to-first-SRE, Time-to-Bone-Metastasis-Progression and Time-to-Visceral-Metastasis-Progression. Conversely, during treatment monitoring, positive ΔAUC value, expression of RANK-positive CTCs persistence, correlated with longer Time-to-first-SRE (p = 0.0002) and Time-to-Bone-Metastasis-Progression (p = 0.0012). Furthermore, the early increase at second day, in RANK-positive CTCs (Positive-Slope) was associated with delay in time-to-first-SRE (p = 0.0038) and Time-to-Bone-Metastasis-Progression (p = 0.0024). We demonstrate, for the first time, the expression of RANK on CTCs in MBC patients and that the persistence of RANK expression determines denosumab effectiveness.
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http://dx.doi.org/10.1038/s41598-020-58339-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6987166PMC
January 2020

Synthesis of Cyclohepta[]indoles by (4 + 3) Cycloaddition of 2-Vinylindoles or 4-Furo[3,2-]indoles with Oxyallyl Cations.

J Org Chem 2020 Mar 6;85(5):3265-3276. Epub 2020 Feb 6.

Dipartimento di Scienze Farmaceutiche-Sezione di Chimica Generale e Organica "A. Marchesini", Università degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy.

The synthesis of cyclohepta[]indole derivatives through the dearomative (4 + 3) cycloaddition reaction of 2-vinylindoles or 4-furo[3,2-]indoles with in situ generated oxyallyl cations is reported. Oxyallyl cations are generated from α-bromoketones in the presence of a base and a perfluorinated solvent. Cyclohepta[]indole scaffolds are obtained under mild reaction conditions, in the absence of expensive catalysts, starting from simple reagents, in good to excellent yields and with complete diasteroselectivity. Preliminary expansion of the scope to 3-vinylindoles and to aza-oxyallyl cations is reported.
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http://dx.doi.org/10.1021/acs.joc.9b03117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997566PMC
March 2020

Evaluation of Passive Immunity Induced by Immunisation Using Two Inactivated gE-deleted Marker Vaccines against Infectious Bovine Rhinotracheitis (IBR) in Calves.

Vaccines (Basel) 2020 Jan 4;8(1). Epub 2020 Jan 4.

National Reference Laboratory for Infectious Bovine Rhinotracheitis (IBR), Istituto Zooprofilattico Sperimentale Umbria-Marche "Togo Rosati", 06126 Perugia, Italy.

Different types of vaccines against Infectious Bovine Rhinotracheitis (IBR) are commercially available. Among these, inactivated glycoprotein E (gE)-deleted marker vaccines are commonly used, but their ability to induce passive immunity is poorly known. Here, we evaluated the passive immunity transferred from dams immunised with commercial inactivated gE-deleted marker vaccines to calves. We vaccinated 12 pregnant cattle devoid of neutralising antibodies against Bovine alphaherpesvirus 1 (BoHV-1) and divided them into two groups with 6 animals each. Both groups were injected with a different inactivated gE-deleted marker vaccine administrated via intranasal or intramuscular routes. An additional 6 pregnant cattle served as the unvaccinated control group. After calving, the number of animals in each group was increased by the newborn calves. In the dams, the humoral immune response was evaluated before calving and, subsequently, at different times until post-calving day 180 (PCD180). In addition, the antibodies in colostrum, milk, and in serum samples from newborn calves were evaluated at different times until PCD180. The results indicated that inactivated glycoprotein E (gE)-deleted marker vaccines are safe and produce a good humoral immune response in pregnant cattle until calving and PCD180. Moreover, results showed that, in calf serum, passive immunity persists until PCD180.
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http://dx.doi.org/10.3390/vaccines8010014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157740PMC
January 2020

Single-Cell Analysis of Circulating Tumor Cells: How Far Have We Come in the -Omics Era?

Front Genet 2019 17;10:958. Epub 2019 Oct 17.

Veneto Institute of Oncology IOV-IRCCS, Padua, Italy.

Tumor cells detach from the primary tumor or metastatic sites and enter the peripheral blood, often causing metastasis. These cells, named Circulating Tumor Cells (CTCs), display the same spatial and temporal heterogeneity as the primary tumor. Since CTCs are involved in tumor progression, they represent a privileged window to disclose mechanisms of metastases, while -omic analyses at the single-cell level allow dissection of the complex relationships between the tumor subpopulations and the surrounding normal tissue. However, in addition to reporting the proof of concept that we can query CTCs to reveal tumor evolution throughout the continuum of treatment for early detection of resistance to therapy, the scientific literature has also been highlighting the disadvantages of CTCs, which hampers a routine use of this approach in clinical practice. To date, an increasing number of CTC technologies, as well as -omics methods, have been employed, mostly lacking strong comparative analyses. The rarity of CTCs also represents a major challenge, because there is no consensus regarding the minimal criteria necessary and sufficient to define an event as CTC; moreover, we cannot often compare data from of one study with that of another. Finally, the availability of an individual tumor profile undermines the traditional histology-based treatment. Applying molecular data for patient benefit implies a collective effort by biologists, bioengineers, and clinicians, to create tools to interpret molecular data and manage precision medicine in every single patient. Herein, we focus on the most recent findings in CTC -omics to learn how far we have come.
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http://dx.doi.org/10.3389/fgene.2019.00958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811661PMC
October 2019

Ground Beetle (Coleoptera: Carabidae) Assemblages and Slug Abundance in Agricultural Fields Under Organic and Low-Input Conventional Management Within a Long-Term Agronomic Trial in Central Italy.

Environ Entomol 2019 12;48(6):1377-1387

CiRAA Centre for Agri-environmental Research 'Enrico Avanzi', San Piero a Grado, Pisa, Italy.

Inside a long-term agronomic trial aimed at evaluating the effects of organic and low-input conventional management systems on soil fertility and arable crop production, we selected six fields bordered by hedgerows, three under each management system. Here, we analyzed the carabid assemblages and the slug abundance. Samplings took place in five different periods, across 1 yr of observations. The carabid abundances were similar in organic and conventional fields. The Shannon-Wiener diversity index (H') showed a higher value in the conventional fields, although in the organic fields, a higher number of species were observed. The multivariate analysis described similar carabid communities, but excluding the period factor, it showed a significant influence of the management system. There was no difference between the captures of traps placed along the hedgerow and in the middle, whereas in the conventional fields, the hedgerow traps captured a higher number of specimens, showing a role of the hedgerow as carabid reservoir. The slugs were present mainly while green manure was grown on the organic fields where also Poecilus cupreus Linné, 1758 (Coleoptera: Carabidae) was captured abundantly.
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http://dx.doi.org/10.1093/ee/nvz119DOI Listing
December 2019

Effects of lipopolysaccharide and juvenile hormone III treatments on cell growth and gene expression in the Ceratitis capitata (Diptera: Tephritidae) CCE/CC128 cell line.

Arch Insect Biochem Physiol 2019 Dec 12;102(4):e21617. Epub 2019 Sep 12.

Department of Zoology and Physical Anthropology, Faculty of Veterinary, Campus Mare Nostrum, University of Murcia, Murcia, Spain.

The Mediterranean fruit fly Ceratitis capitata is one of the most important insect pest species in the world. It has a high colonization capacity and population variety, giving it considerable genetic diversity. Strategies for its control have included the sterile insect technique and insect growth regulators. Many studies have analyzed the medfly transcriptome, and along with the medfly genome sequence, the sequences of multiple genes related to sex determination, mating, development, pheromone detection, immunity, or stress have been identified. In this study, the medfly CCE/CC128 cell line was used to assess cell growth variation and changes in the expression of genes covering different functions, after lipopolysaccharide (LPS) and juvenile hormone III (JHIII) treatments. No significant effects on cell growth and gene expression were observed in the cells treated with LPS. In the cells treated with JHIII, the results showed significant effects on cell growth, and an overexpression was found of the Shade gene, one of the Halloween gene members of the cytochrome p450 family, which is involved in development and the synthesis of 20-hydroxyecdysone. This study shows preliminary results on the insect cell line in combination with whole-genome sequencing, which can facilitate studies regarding growth, toxicity, immunity, and transcriptome regulations as a response to different compounds and environmental alterations.
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http://dx.doi.org/10.1002/arch.21617DOI Listing
December 2019

CTCs 2020: Great Expectations or Unreasonable Dreams.

Cells 2019 08 27;8(9). Epub 2019 Aug 27.

Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, 47014 Meldola, Italy.

Circulating tumor cells (CTCs) are cellular elements that can be scattered into the bloodstream from primary cancer, metastasis, and even from a disseminated tumor cell (DTC) reservoir. CTCs are "seeds", able to give rise to new metastatic lesions. Since metastases are the cause of about 90% of cancer-related deaths, the significance of CTCs is unquestionable. However, two major issues have stalled their full clinical exploitation: rarity and heterogeneity. Therefore, their full clinical potential has only been predicted. Finding new ways of studying and using such tremendously rare and important events can open new areas of research in the field of cancer research, and could drastically improve tumor companion diagnostics, personalized treatment strategies, overall patients management, and reduce healthcare costs.
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http://dx.doi.org/10.3390/cells8090989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769853PMC
August 2019

Genotypic Characterization of Torymus sinensis (Hymenoptera: Torymidae) After Its Introduction in Tuscany (Italy) for the Biological Control of Dryocosmus kuriphilus (Hymenoptera: Cynipidae).

J Insect Sci 2019 Jul;19(4)

Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto, Pisa, Italy.

Torymus sinensis Kamijo (Hymenoptera: Torymidae) is an alien parasitoid that is used in many areas of the world for biological control the Asian chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae). In Italy, this parasitoid was imported from Japan in 2003 and subsequently multiplied and released throughout the country. In this study, a phylogenetic investigation was carried out on insects from three different sites in northern Tuscany (Italy). Moreover, the possible hybridization between T. sinensis and some native Torymus species was evaluated. The conserved region 18S rRNA gene and the hypervariable ITS2 (Internal Transcribed Spacer 2) region of the ribosomal cistrone were selected as molecular markers. Sequencing the amplified products, after cloning, ruled out any hybridization between T. sinensis and the native Torymus species, and also confirmed the presence of two haplotypes for the Tuscan population of T. sinensis both for the region of the 18S rRNA gene as well as for the ITS2 region. These results confirm that the environmental impact of the alien parasitoid T. sinensis in the study site is acceptable, although an extensive and repeated monitoring would be desirable.
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http://dx.doi.org/10.1093/jisesa/iez080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698180PMC
July 2019

Gold-Catalyzed Cascade Reactions of 4 H-Furo[3,2- b]indoles with Allenamides: Synthesis of Indolin-3-one Derivatives.

J Org Chem 2019 May 9;84(9):5150-5166. Epub 2019 Apr 9.

Dipartimento di Scienze Farmaceutiche-Sezione di Chimica Generale e Organica "A. Marchesini" , Università degli Studi di Milano , Via Venezian 21 , 20133 Milano , Italy.

Merging the ability of cationic gold(I) catalysts to activate unsaturated π-systems with the electrophiles-driven ring-opening reactions of furans, we describe a new approach to synthesize 2-spiroindolin-3-ones from 4 H-furo[3,2- b]indoles. The reaction occurs through a cascade sequence involving addition of a gold-activated allene to the furan moiety of the starting furoindole followed by a ring-opening/ring-closing event affording 2-spirocyclopentane-1,2-dihydro-3 H-indolin-3-ones in moderate to good yields.
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http://dx.doi.org/10.1021/acs.joc.9b00143DOI Listing
May 2019

Detection and Prognostic Relevance of Circulating and Disseminated Tumour Cell in Dogs with Metastatic Mammary Carcinoma: A Pilot Study.

Cancers (Basel) 2019 Feb 1;11(2). Epub 2019 Feb 1.

Istituto Zooprofilattico Sperimentale delle Venezie, 35020 Legnaro (PD), Italy.

In human breast cancer, both circulating tumour cells (CTCs) in peripheral blood and disseminated tumour cells (DTCs) in the bone marrow are predictive of short survival and may be used as liquid biopsy to guide therapy. Herein we investigate, for the first time, the feasibility to quantify CTCs and DTCs in canine metastatic mammary carcinoma (MMC) with the automated CellSearch platform, which identifies tumour cells by immune-magnetic enrichment and fluorescent labelling. Using this approach before start of treatment, we could detect at least 1 CTC per 7.5 mL of peripheral blood in 12 out of 27 evaluable samples (44.4%) and at least 1 DTC per 1 mL of bone marrow in 11 out of 14 evaluable samples (78.6%). Conversely, we did not find any CTCs in the healthy, negative control dogs ( = 5) that we analysed in parallel. Interestingly, the levels of CTCs/DTCs and the prevalence of positive dogs closely resemble results obtained by CellSearch assay in metastatic breast cancer patients at diagnosis. Moreover, in the canine cohort, the presence of CTCs was significantly associated with poor outcome. These observations identify the first actionable marker in veterinarian oncology to guide treatment of canine MMC. Furthermore, our findings have important implications for human research, since it reinforce the value of canine MMC as model useful to speed up pharmacological studies with primary endpoint of overall survival, given the reduced life-span of the canine species.
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http://dx.doi.org/10.3390/cancers11020163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406716PMC
February 2019

Single tube liquid biopsy for advanced non-small cell lung cancer.

Int J Cancer 2019 06 28;144(12):3127-3137. Epub 2019 Jan 28.

Department of Pulmonary Diseases, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands.

The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing. We studied the relation between overall survival (OS) and the presence of four cancer biomarkers from a single blood draw in advanced NSCLC patients: EpCAM circulating tumor cells (CTC), EpCAM CTC, tumor-derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA). EpCAM CTC were detected with CellSearch, tdEV in the CellSearch images and EpCAM CTC with filtration after CellSearch. ctDNA was isolated from plasma and mutations present in the primary tumor were tracked with deep sequencing methods. In 97 patients, 21% had ≥2 EpCAM CTC, 15% had ≥2 EpCAM CTC, 27% had ≥18 tdEV and 19% had ctDNA with ≥10% mutant allele frequency. Either one of these four biomarkers could be detected in 45% of the patients and all biomarkers were present in 2%. In 11 out of 16 patients (69%) mutations were detected in the ctDNA. Two or more unfavorable biomarkers were associated with poor OS. The presence of EpCAM CTC and elevated levels of tdEV and ctDNA was associated with a poor OS; however, the presence of EpCAM CTC was not. This single tube approach enables simultaneous analysis of multiple biomarkers to explore their potential as a liquid biopsy.
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http://dx.doi.org/10.1002/ijc.32056DOI Listing
June 2019

EpCAM and EpCAM circulating tumor cells in metastatic prostate and breast cancer patients.

Oncotarget 2018 Nov 2;9(86):35705-35716. Epub 2018 Nov 2.

Department of Medical Cell BioPhysics, Faculty of Sciences and Technology, MIRA Institute, University of Twente, Enschede, The Netherlands.

The presence of high expressing epithelial cell adhesion molecule (EpCAM) circulating tumor cells (CTC) enumerated by CellSearch in blood of cancer patients is strongly associated with poor prognosis. This raises the question about the presence and relation with clinical outcome of low EpCAM expressing CTC (EpCAM CTC). In the EU-FP7 CTC-Trap program, we investigated the presence of EpCAM and EpCAM CTC using CellSearch, followed by microfiltration of the EpCAM CTC depleted blood. Blood samples of 108 castration-resistant prostate cancer patients and 22 metastatic breast cancer patients were processed at six participating sites, using protocols and tools developed in the CTC-Trap program. Of the prostate cancer patients, 53% had ≥5 EpCAM CTC and 28% had ≥5 EpCAM CTC. For breast cancer patients, 32% had ≥5 EpCAM CTC and 36% had ≥5 EpCAM CTC. 70% of prostate cancer patients and 64% of breast cancer patients had in total ≥5 EpCAM and/or EpCAM CTC, increasing the number of patients in whom CTC are detected. Castration-resistant prostate cancer patients with ≥5 EpCAM CTC had shorter overall survival versus those with <5 EpCAM CTC ( = 0.000). However, presence of EpCAM CTC had no relation with overall survival. This emphasizes the importance to demonstrate the relation with clinical outcome when presence of CTC identified with different technologies are reported, as different CTC subpopulations can have different relations with clinical outcome.
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http://dx.doi.org/10.18632/oncotarget.26298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235023PMC
November 2018

Clonal heterogeneity of melanoma in a paradigmatic case study: future prospects for circulating melanoma cells.

Melanoma Res 2019 02;29(1):89-94

Veneto Institute of Oncology IOV - IRCCS, Padua, Italy.

The management of metastatic melanoma is a difficult matter. Nevertheless, the advent of target therapy has significantly improved patient outcome, provided that tumor molecular characteristics become available: the detection of drug-resistant clones can contribute to understanding the reasons for resistance onset, influencing the choice of subsequent therapy. This work aimed to provide a possible explanation for the early resistance to vemurafenib developed by a patient with melanoma, and concurrently to assess the extent, and role, of the tumor clonal heterogeneity. We analyzed tissue samples from different sites and time points: first/second primary, three lymph node metastases, and circulating melanoma cells (CMCs). We first investigated these samples by the routine Sanger sequencing for BRAF, NRAS, and KIT, and then, we focused on specific hotspots by droplet digital PCR. We detected a BRAF V600E mutation by Sanger sequencing in the second primary and distant lymph node metastases, but not in the first primary or sentinel lymph node. Interestingly, by droplet digital PCR, the V600E mutation was also detected in the first primary, and the V600K in the second primary and metastases. Moreover, we identified a rare KIT V569G mutation, appearing to be CMC exclusive. This finding confirms the potential of CMCs as a source of tumor material for genetic analysis, reflecting real-time systemic disease evolution and, most likely, the most aggressive, treatment-resistant clones. In summary, this work underlines the importance of CMCs in the early identification of tumor clones putatively responsible for therapy resistance.
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http://dx.doi.org/10.1097/CMR.0000000000000510DOI Listing
February 2019

What information could the main actors of liquid biopsy provide? -a representative case of non-small cell lung cancer (NSCLC).

J Thorac Dis 2018 Jul;10(7):E570-E576

IOV-IRCCS, Padova, Italy.

In non-small cell lung cancer (NSCLC), there is a consensus regarding the use of liquid biopsy, generally, to detect "druggable" mutations and, in particular, to monitor tyrosine kinase inhibitor (TKI) treatments. However, whether circulating tumor cells (CTCs) are better tools than cell-free DNA (cfDNA), is still a matter of debate, mainly concerning which antigen(s) we should use to investigating simultaneously both epithelial and epithelial-to-mesenchymal transient (EMT) phenotype in the same sample of CTCs. To address this item, we exploited here a single-tube liquid biopsy, to detect both epithelial cell adhesion molecule (EpCAM)-positive CTCs and EpCAM-low/negative CTCs, because down-modulation of EpCAM is considered the first step in EMT. Furthermore, we analyzed the DNA from CTCs of four different phenotypes (ctcDNA), according to their EpCAM expression and cytokeratin pattern, and circulating tumor DNA (ctDNA) by droplet digital PCR (ddPCR), in order to disclose activating and resistance-driving mutations. Liquid biopsy reflected spatial and temporal heterogeneity of the tumor under treatment pressure. We provide the proof-of-concept that the complementary use of ctDNA and ctcDNA represents a reliable, minimally invasive and dynamic tool for a more comprehensive view of tumor evolution.
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http://dx.doi.org/10.21037/jtd.2018.06.38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105979PMC
July 2018

Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap).

Int J Cancer 2018 11 19;143(10):2584-2591. Epub 2018 Sep 19.

Prostate Cancer Targeted Therapies Group, The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research, Sutton, UK.

Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a "liquid biopsy". In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as "gold standard" reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0-32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 μm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1-4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.
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http://dx.doi.org/10.1002/ijc.31752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637919PMC
November 2018