Towards a correlative approach for characterising single virus particles by transmission electron microscopy and nanoscale Raman spectroscopy.

The morphology and structure of biological nanoparticles, such as viruses, can be efficiently analysed by transmission electron microscopy (TEM). To chemically characterise such nanoparticles in heterogeneous samples at the single particle level, we suggest tip-enhanced Raman spectroscopy (TERS) as a correlative method. Here we describe a TERS-compatible staining procedure for TEM which involves sample pre-scanning by TEM imaging, nanoparticle relocalisation by atomic force microscopy (AFM) followed by spectroscopic characterization of the virus nanoparticles using TERS. First successful correlative measurements are demonstrated on tobacco mosaic virus particles deposited on silicon-based TEM sample supports. In addition, the advantages and problems of this methodology are discussed.

Vaccination with HPV16 peptides of patients with advanced cervical carcinoma: clinical evaluation of a phase I-II trial.

A phase I-II clinical trial was performed involving vaccination with HPV16 E7 peptides of patients suffering from HPV16 positive cervical carcinoma which was refractory to conventional treatment. Patients receiving the vaccine were HLA-A*0201 positive with HPV16 positive cervical carcinoma. The clinical trial was designed as a dose-escalation study, in which successive groups of patients received 100 micrograms, 300 micrograms or 1000 micrograms of each peptide, respectively. The vaccine consisted of two HPV16 E7 peptides and one helper peptide emulsified in Montanide ISA 51 adjuvant. 19 patients were included in the study, no adverse side-effects were observed. 2 patients showed stable disease for 1 year after vaccination; 15 patients showed progressive disease of whom 1 died during the vaccination treatment due to progressive disease; and 2 patients showed tumour-regression after chemotherapy following vaccination. A relative low count of lymphocytes before and after vaccination was present in 11/19 patients indicating that these patients were immunocompromised. This study shows that HPV16 E7 peptide vaccination is feasible, even in a group of patients with terminal disease. This paves the way for vaccinating patients with less advanced disease, whose immune system is less compromised by progressive disease.

Discrimination of distinct subpopulations within a tumor with combined double immunophenotyping and interphase cytogenetics.

We describe a method that enables detection and immunophenotypical characterization of distinct subpopulations within a cytogenetically defined tumor clone. Coexisting normal cells do not hinder microscopic evaluation because they can be distinguished from cytogenetically aberrant tumor cells. This is also true when normal and neoplastic cells cannot be clearly distinguished by cytology or immunohistochemistry, i.e., if both constituents have similar immunophenotypes and morphology. The method is based on fluorescence double staining for two different antigens combined with interphase cytogenetic analysis. It is referred to as "Fluorescence immunophenotyping and Interphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)." In a case of follicular lymphoma we demonstrate that FICTION can differentiate bcl-2-positive malignant and non-malignant cells and can verify the presence of bcl-2-positive but cytogenetically inconspicuous T-lymphocytes.

Rationalization of in situ hybridization: testing up to 16 different probes on a single slide.

Although particularly interested in tumor research, investigators in many tumor cytogenetics laboratories cannot afford extensive molecular/interphase cytogenetics studies in addition to routine cytogenetics analyses, primarily because many slides must be stained to examine a few cases using a panel of centromeric probes. Moreover, fluorescence in situ hybridization (FISH) techniques mostly require freshly prepared reagents, such as hybridization mixtures or antibody solutions. These technical requirements are very time-consuming, thus limiting their use in routine screening. We report a significantly economical method for rapid performance of interphase cytogenetics in great numbers of cases. The major advantage if its superior efficiency: up to 16 different probes may be used on one single slide. Moreover, the method is significantly cheaper than other techniques owing to minimal probe consumption. The technique is also best suited if great numbers of new probes, e.g., polymerase chain reaction (PCR)-generated YAC-probes, must be tested for their applicability in FISH.

Rapid immunophenotypic characterization of chromosomally aberrant cells by the new FICTION method.

Recently, we have presented a new technique for immunophenotyping cells that have numerical chromosome aberrations. We referred to this method as "Fluorescence-Immunophenotyping and Interphase Cytogenetics as a Tool for Investigation of Neoplasms" (FICTION). We present here an advanced FICTION method with three-color staining and improved sensitivity.

Simultaneous fluorescence immunophenotyping and interphase cytogenetics: a contribution to the characterization of tumor cells.

In immunocytochemical studies, the phenotypic evaluation of tumor cells is often complicated by accompanying normal cells, representing the original tissue or infiltrating leukocytes. This holds particularly true for tissues with a great morphological and immunophenotypical variability, such as bone marrow. A method that identifies mitotic tumor cells by chromosomal aberrations and permits the subsequent immunophenotypical analysis was a first progress, demonstrated by Teerenhovi et al. However, the results are usually hampered by the low number of analyzable mitoses. We demonstrate here a method that simultaneously combines immunophenotyping and in situ hybridization with centromere-specific probes. Using our method, numerically aberrant tumor cells can be identified by interphase cytogenetics and subsequently analyzed immunophenotypically. Since all interphase cells can be analyzed, we are not limited by the number and banding quality of analyzable mitoses.