Use of Formalin-Fixed Paraffin-Embedded Samples for Gene Expression Studies in Breast Cancer Patients

To obtain gene expression profiles from samples collected in clinical trials, we conducted a pilot study to assess feasibility and estimate sample attrition rates when profiling formalin-fixed, paraffin-embedded specimens. Ten matched fresh-frozen and fixed breast cancer samples were profiled using the Illumina HT-12 and Ref-8 chips, respectively. The profiles obtained with Ref 8, were neither technically nor biologically reliable since they failed to yield the expected separation between estrogen receptor positive and negative samples. With the use of Affymetrix HG-U133 2.0 Plus chips on fixed samples and a quantitative polymerase chain reaction -based sample pre-assessment step, results were satisfactory in terms of biological reliability, despite the low number of present calls (M = 21%±5). Compared with the Illumina DASL WG platform, Affymetrix data showed a wider interquartile range (1.32 vs 0.57, P<2.2 E-16,) and larger fold changes. The Affymetrix chips were used to run a pilot study on 60 fixed breast cancers. By including in the workflow the sample pre-assessment steps, 96% of the samples predicted to give good results (44/46), were in fact rated as satisfactory from the point of view of technical and biological meaningfulness. Our gene expression profiles showed strong agreement with immunohistochemistry data, were able to reproduce breast cancer molecular subtypes, and allowed the validation of an estrogen receptor status classifier derived in frozen samples. The approach is therefore suitable to profile formalin-fixed paraffin-embedded samples collected in clinical trials, provided that quality controls are run both before (sample pre-assessment) and after hybridization on the array.     

On the first record of the genus Octopoteuthis (Cephalopoda: Octopoteuthidae) in the Sardinian Channel (central western Mediterranean Sea)

A specimen of the rare squid genus Octopoteuthis (Cephalopoda: Octopoteuthidae) from 1600 m in the Sardinian Channel (central western Mediterranean) is described. Meristic and morphological data are provided. The specimen was a spent female and details of the reproductive system and implanted spermatangia are reported. Genetic analysis of this specimen and comparison with those available for species in this genus were carried out. Due to issues associated with the taxonomy of the genus Octopoteuthis, it was not possible to identify the specimen to the species level. The taxonomy of the genus is discussed.     

Analysis of hepatitis C virus hypervariable region 1 sequence from cryoglobulinemic patients and associated controls

Chronic hepatitis C virus (HCV) infection is frequently associated with extrahepatic manifestations, including nonmalignant and malignant B-cell lymphoproliferative disorders. It has been reported that specific changes or recurring motifs in the amino acid sequence of the HCV hypervariable region 1 (HVR1) may be associated with cryoglobulinemia. We searched for specific insertions/deletions and/or amino acid motifs within HVR1 in samples from 80 symptomatic and asymptomatic patients with and 33 patients without detectable cryoglobulins, all with chronic HCV infection. At variance with the results of a previous study which reported a high frequency of insertions at position 385 of HVR1 from cryoglobulinemic patients, we found a 6.2% prevalence of insertions in samples from patients with and a 9.1% prevalence in those without cryoglobulinemia. Moreover, statistical and bioinformatics approaches including Fisher's exact test, k-means clustering, Tree determinant-residue identification, correlation of mutations, principal component analysis, and phylogenetic analysis failed to show statistically significant differences between sequences from cryoglobulin-negative and -positive patients. Our findings suggest that cryoglobulinemia may arise by virtue of as-yet-unidentified host- rather than virus-specific factors. Specific changes in HCV envelope sequence distribution are unlikely to be directly involved in the establishment of pathological B-cell monoclonal proliferation.     

Water quality and diversity of the Recent ostracod fauna in lowland springs from Lombardy (northern Italy)

Sedimentary records provide important information for understanding changes in the history of eutrophication in Lake Taihu. In addition, the catchment nutrient model SWAT provides a powerful tool to examine eutrophic changes in a long-term context. Since it is difficult to evaluate impacts of natural eutrophic development and anthropogenic changes in catchment discharge and land use, simulation of past changes provides a mirror on processes and dynamics. Boundaries in the simulations are set to a pre-industrial time to evaluate natural-agricultural nutrient changes in Taihu Basin a 100 years ago. Total nitrogen (TN) and total phosphorus (TP) in the main channel flowing into the lake are simulated in four sub-basins for 200 model years. Results show that modeling can capture basic features of basin nutrient development, where mean TN concentration (0.12 mg l⁻¹) can be compared in broad scale to mean TN concentration (0.17 mg kg⁻¹) from Lake Taihu sedimentary cores dating back about 100 years. Spatial nutrient simulations suggest that the two major nutrient sources are from the southwestern sub-basin (48% TN and 68% TP of the basin total) and the northwestern sub-basin (18% TN and 17% TP). There are differences of +7.3 × 10⁴ kg TN and +2.0 × 10⁵ kg TP between total input and output values, simulating mean annual amounts of nutrient deposited into the lake. TN and TP concentration differences between input and output sub-basins become smaller in the second 100 years than the first 100 years, suggesting a 100 year period to reach a balance of net nutrients. Catchment nutrient modeling provides a basis to evaluate how nutrient production and balance responded to environmental changes over 200 years in Taihu Basin.     

Insertion of a magnesium(II)-octacarboranyl(hexylsulfanyl) porphyrazine into liposomes: a physico-chemical study

The synthesis, characterization and liposome insertion of a novel magnesium(II) carboranyl-porphyrazine, i.e. [2,3,7,8,12,13,17,18-octakis-(1,2-dicarba-closo-dodecaboranyl)-hexylthio-5,10,15,20-porphyrazine]magnesium(II) complex, MgHECSPz, are described. MgHECSPz was designed to improve the potentiality in multiple approach anticancer therapy. Liposomal formulations with different surface charge were prepared as delivering agents. The obtained loaded vectors were characterized by DLS, SAXS, SANS and zeta potential measurements in order to define the overall properties and structural details of loaded liposomes.     

Lysine 58-cleaved beta2-microglobulin is not detectable by 2D electrophoresis in ex vivo amyloid fibrils of two patients affected by dialysis-related amyloidosis

The lysine 58 cleaved and truncated variant of beta(2)-microglobulin (DeltaK58-beta2m) is conformationally unstable and present in the circulation of a large percentage of patients on chronic hemodialysis, suggesting that it could play a role in the beta2-microglobulin (beta2m) amyloid fibrillogenesis associated with dialysis-related amyloidosis (DRA). However, it has yet to be detected in the amyloid deposits of such patients. Here, we extracted amyloid fibrils, without denaturation or additional purification, from different amyloidotic tissues of two unrelated individuals suffering from DRA, and characterized them by high-sensitivity bidimensional gel electrophoresis (2D-PAGE), immunoblotting, MALDI time-of-flight mass spectrometry, and protein sequencing. To confirm whether or not this species could be identified by our proteomic approaches, we mapped its location in 2D-PAGE, in mixtures of pure DeltaK58-beta2m, and extracts of amyloid fibrils from patients, to a discrete region of the gel distinct from other isoforms of beta2m. Using this approach, the two known principal isoforms found in beta2m amyloid were identified, namely, the full-length protein and the truncated species lacking six N-terminal amino acid residues (DeltaN6-beta2m). In contrast, we found no evidence for the presence of DeltaK58-beta2m.     

Assessment of EGFR Mutations in Circulating Tumor Cell Preparations from NSCLC Patients by Next Generation Sequencing: Toward a Real-Time Liquid Biopsy for Treatment

Introduction

Assessment of EGFR mutation in non-small cell lung cancer (NSCLC) patients is mandatory for optimization of pharmacologic treatment. In this respect, mutation analysis of circulating tumor cells (CTCs) may be desirable since they may provide real-time information on patient''s disease status.

Experimental Design

Blood samples were collected from 37 patients enrolled in the TRIGGER study, a prospective phase II multi-center trial of erlotinib treatment in advanced NSCLC patients with activating EGFR mutations in tumor tissue. 10 CTC preparations from breast cancer patients without EGFR mutations in their primary tumors and 12 blood samples from healthy subjects were analyzed as negative controls. CTC preparations, obtained by the Veridex CellSearch System, were subjected to ultra-deep next generation sequencing (NGS) on the Roche 454 GS junior platform.

Results

CTCs fulfilling all Veridex criteria were present in 41% of the patients examined, ranging in number between 1 and 29. In addition to validated CTCs, potential neoplastic elements were seen in 33 cases. These included cells not fulfilling all Veridex criteria (also known as “suspicious objects”) found in 5 (13%) of 37 cases, and isolated or clustered large naked nuclei with irregular shape observed in 33 (89%) cases. EGFR mutations were identified by NGS in CTC preparations of 31 (84%) patients, corresponding to those present in matching tumor tissue. Twenty-five (96%) of 26 deletions at exon 19 and 6 (55%) of 11 mutations at exon 21 were detectable (P = 0.005). In 4 (13%) cases, multiple EGFR mutations, suggesting CTC heterogeneity, were documented. No mutations were found in control samples.

Conclusions

We report for the first time that the CellSearch System coupled with NGS is a very sensitive and specific diagnostic tool for EGFR mutation analysis in CTC preparations with potential clinical impact.     

Reporting Tumor Molecular Heterogeneity in Histopathological Diagnosis

Background

Detection of molecular tumor heterogeneity has become of paramount importance with the advent of targeted therapies. Analysis for detection should be comprehensive, timely and based on routinely available tumor samples.

Aim

To evaluate the diagnostic potential of targeted multigene next-generation sequencing (TM-NGS) in characterizing gastrointestinal cancer molecular heterogeneity.

Methods

35 gastrointestinal tract tumors, five of each intestinal type gastric carcinomas, pancreatic ductal adenocarcinomas, pancreatic intraductal papillary mucinous neoplasms, ampulla of Vater carcinomas, hepatocellular carcinomas, cholangiocarcinomas, pancreatic solid pseudopapillary tumors were assessed for mutations in 46 cancer-associated genes, using Ion Torrent semiconductor-based TM-NGS. One ampulla of Vater carcinoma cell line and one hepatic carcinosarcoma served to assess assay sensitivity. TP53, PIK3CA, KRAS, and BRAF mutations were validated by conventional Sanger sequencing.

Results

TM-NGS yielded overlapping results on matched fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues, with a mutation detection limit of 1% for fresh-frozen high molecular weight DNA and 2% for FFPE partially degraded DNA. At least one somatic mutation was observed in all tumors tested; multiple alterations were detected in 20/35 (57%) tumors. Seven cancers displayed significant differences in allelic frequencies for distinct mutations, indicating the presence of intratumor molecular heterogeneity; this was confirmed on selected samples by immunohistochemistry of p53 and Smad4, showing concordance with mutational analysis.

Conclusions

TM-NGS is able to detect and quantitate multiple gene alterations from limited amounts of DNA, moving one step closer to a next-generation histopathologic diagnosis that integrates morphologic, immunophenotypic, and multigene mutational analysis on routinely processed tissues, essential for personalized cancer therapy.