The pre-analytical phase of the liquid biopsy.

The term 'liquid biopsy', introduced in 2013 in reference to the analysis of circulating tumour cells (CTCs) in cancer patients, was extended to cell-free nucleic acids (cfNAs) circulating in blood and other body fluids. CTCs and cfNAs are now considered diagnostic and prognostic markers, used as surrogate materials for the molecular characterisation of solid tumours, in particular for research on tumour-specific or actionable somatic mutations. Molecular characterisation of cfNAs and CTCs (especially at the single cell level) is technically challenging, requiring highly sensitive and specific methods and/or multi-step processes.

Analytical Evaluation of an NGS Testing Method for Routine Molecular Diagnostics on Melanoma Formalin-Fixed, Paraffin-Embedded Tumor-Derived DNA.

Next Generation Sequencing (NGS) is a promising tool for the improvement of tumor molecular profiling in view of the identification of a personalized treatment in oncologic patients. To verify the potentiality of a targeted NGS (Ion AmpliSeq™ Cancer Hotspot Panel v2), selected melanoma samples ( = 21) were retrospectively analyzed on S5 platform in order to compare NGS performance with the conventional techniques adopted in our routine clinical setting (Sequenom MassARRAY system, Sanger sequencing, allele-specific real-time PCR). The capability in the identification of rare and low-frequency mutations in the main genes involved in melanoma ( and genes) was verified and integrated with the results deriving from other oncogenes and tumor suppressor genes.

Personalized laboratory medicine: a patient-centered future approach.

In contrast to population-based medical decision making, which emphasizes the use of evidence-based treatment strategies for groups of patients, personalized medicine is based on optimizing treatment at the level of the individual patient. The creation of molecular profiles of individual patients was made possible by the advent of "omics" technologies, based on high throughput instrumental techniques in combination with biostatistics tools and artificial intelligence. The goal of personalized laboratory medicine is to use advanced technologies in the process of preventive, curative or palliative patient management.

Histone 3.3 mutations in giant cell tumor and giant cell-rich sarcomas of bone.

Mutually exclusive histone 3.3 gene mutations have been recognized in chondroblastoma and giant cell tumor of bone (GCTB), which may be useful for differential diagnostic purposes in morphologically ambiguous cases. Although more than 90% of GCTBs present histone 3.

Phenotypic and molecular differences between giant-cell tumour of soft tissue and its bone counterpart.

Aims: Giant-cell tumour (GCT) of soft tissue (GCT-ST) is a primary soft tissue neoplasm that is histologically similar to GCT of bone (GCT-B). Recently, it has been reported that >90% of GCT-Bs have a driver mutation in the H3F3A gene. As the relationship between GCT-ST and GCT-B is unclear, the aim of this study was to compare a series of GCT-STs and GCT-Bs with regard to the presence of H3F3A mutations and several immunophenotypic markers.

Mutational analysis of single circulating tumor cells by next generation sequencing in metastatic breast cancer.

Circulating Tumor Cells (CTCs) represent a "liquid biopsy" of the tumor potentially allowing real-time monitoring of cancer biology and therapies in individual patients.The purpose of the study was to explore the applicability of a protocol for the molecular characterization of single CTCs by Next Generation Sequencing (NGS) in order to investigate cell heterogeneity and provide a tool for a personalized medicine approach.CTCs were enriched and enumerated by CellSearch in blood from four metastatic breast cancer patients and singularly isolated by DEPArray.

Denosumab treated giant cell tumour of bone: a morphological, immunohistochemical and molecular analysis of a series.

Aims: Denosumab, a fully human monoclonal antibody directed against RANKL, has recently been introduced in the treatment strategy of giant cell tumour of bone (GCTB). Aim of this study was to investigate the phenotypical modifications induced by denosumab treatment in a series of 15 GCTB.

Methods: The tumours were characterised for histone 3.

Implementation of a companion diagnostic in the clinical laboratory: the BRAF example in melanoma.

A companion diagnostic test provides information that is essential for the safe and effective use of a corresponding therapeutic product as indicated in the drug instructions. The implementation of a companion diagnostic follows the rules of a molecular test for somatic mutations in a routine clinical laboratory environment and needs guidance on practical aspects, including the choice of the proper analytical method and the procedures for internal and external quality controls. Selection of the appropriate assay for detection of genetic alterations depends on several factors: the type of mutation under study, the sample to be assayed and its preparation procedure.

KIT genetic alterations in anorectal melanomas.

Background: Mucosal melanomas (MM) represent a heterogeneous tumour population that exhibits site-specific molecular profiles.

Aims: In a multicentre retrospective study, we investigated KIT aberrations in primary anorectal (AR) melanomas compared with melanoma metastatic to the gastrointestinal (GI) tract.

Methods: Primary AR MM (n=31) and GI metastatic melanoma (n=27) were studied for KIT mutations on exons 11, 13, 17 and 18 by high-resolution melting analysis, direct sequencing and c-KIT expression by immunohistochemistry.

SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA.

Immunohistochemistry is highly sensitive and specific for the detection of NRASQ61R mutation in melanoma.

Testing for NRAS is now integral part in the assessment of metastatic melanoma patients because there is evidence that NRAS-mutated patients may be sensitive to MEK inhibitors, and RAS mutation is a common mechanism of acquired resistance during treatment with BRAF inhibitors. This study evaluated the sensitivity and specificity of immunohistochemical analysis using an N-Ras (Q61R) antibody to detect the presence of the NRASQ61R mutation in melanoma patients. A total of 98 primary cutaneous melanomas that have undergone examination of NRAS mutation were retrieved from a multicentric database.

BRAF and KIT somatic mutations are present in amelanotic melanoma.

The genotypic profile of rare amelanotic melanomas (AMs) has been poorly investigated, thus preventing either an accurate identification as a distinctive melanoma subtype or therapy stratification. Here, we investigated the presence of the BRAF(V600E) mutation by real-time quantitative PCR and KIT mutations (exons 11 and 17) by sequencing analysis in 33 AMs. AMs included 'truly' amelanotic lesions (n = 19), with no melanin pigmentation upon dermoscopic inspection and hypomelanotic lesions (n = 14), by definition partially pigmented lesions showing a melanin pigmentation area of less than 25% of the total surface area.

The somatic affairs of BRAF: tailored therapies for advanced malignant melanoma and orphan non-V600E (V600R-M) mutations.

BRAF V600R-M-D are uncommon mutations, not included in the experimental protocols of BRAF selective inhibitors. We report the evaluation of correlations among different types of BRAF somatic mutations in melanoma and their management with BRAF inhibitors. 21 patients with BRAF mutated metastatic melanoma were enrolled in the protocol with BRAF inhibitors for compassionate use at the University of Modena.

Monitoring the susceptibility to oseltamivir of Influenza A(H1N1) 2009 virus by nested-PCR and pyrosequencing during the pandemic and in the season 2010-2011.

For the early detection of the H275Y mutation as a marker of oseltamivir resistance in A(H1N1) pandemic strains, a sensitive and specific pyrosequencing assay was developed. This assay analyses a region 99nts long, encompassing the H275Y site, amplified by a nested PCR. Seventy-five respiratory specimens, obtained from 62 patients during the pandemic and in the 2010-2011 influenza season, in Tuscany, were tested.

Genetic variants in miR-146a, miR-149, miR-196a2, miR-499 and their influence on relative expression in lung cancers.

Background: The presence of sequence variants in miRNA genes may influence their processing, expression and binding to target mRNAs. Since single miRNA can have a large number of potential mRNA targets, even minor variations in its expression can have influences on hundreds of putative mRNAs.

Methods: Here, we evaluated 101 paired samples (cancer and normal tissues) from non-small cell lung carcinoma (NSCLC) patients to study the genotype distribution of single nucleotide polymorphisms (SNPs) in miR-146a (rs2910164 C-G), miR-149 (rs2292832 C-T), miR-196a2 (rs11614913 C-T) and miR-499 (rs3746444 G-A) and their influence on the expression of respective miRNAs.

High-resolution melting analysis: a new molecular approach for the early detection of Diplodia pinea in Austrian pine.

The differentiation of Diplodia pinea from closely related species, such as Diplodia scrobiculata and Diplodia seriata, and its detection in plant tissue, represented a critical issue for a long time. Molecular screening tools have recently been developed to address this topic. In this study we applied one of the most sensitive and rapid diagnostic screening method so far developed, called High-Resolution Melting Analysis (HRMA), to detect D.

Association between single nucleotide polymorphisms in the XRCC1 and RAD51 genes and clinical radiosensitivity in head and neck cancer.

Purpose: Individual variability in radiosensitivity is large in cancer patients. Single nucleotide polymorphisms (SNPs) in genes involved in DNA repair and in protection against reactive oxygen species (ROS) could be responsible for such cases of radiosensitivity. We investigated the association between the occurrence of acute reactions in 101 patients with squamous cell carcinoma of the head and neck (SCCHN) after radiotherapy (RT) and five genetic polymorphisms: XRCC1 c.

From clinical trials to clinical practice: predictors of response to erlotinib in advanced non-small cell lung cancer patients pretreated with chemotherapy.

Background: Inhibition of the epidermal growth factor receptor pathway with tyrosine kinase inhibitors can improve outcome of patients with advanced non-small cell lung cancer after first-line chemotherapy. The use of clinical characteristics and molecular markers may permit the identification of patients who are more likely to benefit from erlotinib.

Patients And Methods: Retrospective analysis of unselected patients with metastatic non-small cell lung cancer who had previously failed on at least one line of chemotherapy and treated at our institution with erlotinib (150 mg/day orally) until disease progression.

Atypical Spitzoid melanocytic tumors: a morphological, mutational, and FISH analysis.

Background: Identification of the clinical behavior of atypical Spitzoid tumors with conflicting histopathologic features remains controversial.

Objective: We sought to assess whether molecular findings may be helpful in the diagnostic and prognostic assessment of atypical Spitzoid tumors.

Methods: A total of 38 controversial, atypical Spitzoid lesions (≥ 1 mm in thickness) were analyzed for clinicopathological features, chromosomal alterations by fluorescence in situ hybridization (FISH) analysis (RREB1/MYB/CCND1/CEP6), BRAF(V600E) mutation by allele-specific real-time polymerase chain reaction confirmed by sequencing, and H-RAS gene mutation by direct sequencing.

BRAFV600E detection in melanoma is highly improved by COLD-PCR.

Background: The BRAF gene has been identified as an oncogene in human cancer and the V600E mutation has been shown to be associated with clinico pathological features of primary invasive melanomas. As BRAF may be an attractive therapeutic target, it is crucial to have a sensitive method for detecting mutated DNA in biological samples. Our aim was to investigate COLD-PCR (co-amplification at lower denaturation temperature-PCR) as a new approach for the pre-analytical enrichment of the BRAFV600E variant in formalin fixed paraffin embedded (FFPE) melanoma tissues.

The use of COLD-PCR and high-resolution melting analysis improves the limit of detection of KRAS and BRAF mutations in colorectal cancer.

Fast and reliable tests to detect mutations in human cancers are required to better define clinical samples and orient targeted therapies. KRAS mutations occur in 30-50% of colorectal cancers (CRCs) and represent a marker of clinical resistance to cetuximab therapy. In addition, the BRAF V600E is mutated in about 10% of CRCs, and the development of a specific inhibitor of mutant BRAF kinase has prompted a growing interest in BRAF (V600E) detection.

Seladin-1 expression is regulated by promoter methylation in adrenal cancer.

Background: Seladin-1 overexpression exerts a protective mechanism against apoptosis. Seladin-1 mRNA is variably expressed in normal human tissues. Adrenal glands show the highest levels of seladin-1 expression, which are significantly reduced in adrenal carcinomas (ACC).