Immunohistochemical evaluation of HER-2/neu expression in infiltrating breast carcinoma: a study of reproducibility

OBJECTIVE: To determine interobserver and intraobserver reproducibility in the assessment of the HercepTest- and TAB250-immunostained slides. STUDY DESIGN: Three independent expert pathologists (two with and one without training in HercepTest assessment) evaluated the HercepTest and TAB250-immunostained slides of 108 infiltrating breast carcinomas with a triple-blind method. The evaluation was repeated, with the same method and sequence of view, after 60 days. RESULTS: Expert pathologists, after adequate training in HercepTest evaluation, could reach excellent interobserver (K=.911, P<.001) and intraobserver reproducibility (K of .863-.926; P <.001 for all). The percentage of disagreement in intraobserver reproducibility ranged from 0.9% to 3.7%. Interobserver and intraobserver reproducibility in the evaluation of TAB250-immunostained slides was good (K = .658, P < .001) and from good to excellent (K of .600-.895, P < .001 for all), respectively. CONCLUSION: Optimization of the level of accuracy in HercepTest evaluation is mandatory because the decision to initiate therapy with Herceptin depends on the result. Moreover, considering that the percentage of disagreement in intraobserver reproducibility ranges from 0.9% to 3.7%, it is advisable that two expert pathologists evaluate all HercepTest slides with a double-blind method. If there are discordant results, they must be discussed by the same pathologists.     

During apoptosis of tumor cells HMGA1a protein undergoes methylation: identification of the modification site by mass spectrometry

Programmed cell death is characterized by posttranslational modifications of a limited and specific set of nuclear proteins. We demonstrate that during apoptosis of different types of tumor cells there is a monomethylation of the nuclear protein HMGA1a that is associated to its previously described hyperphosphorylation/dephosphorylation process. HMGA1a methylation is strictly related to the execution of programmed cell death and is a massive event that involves large amounts of the protein. In some tumor cells, HMGA1a protein is already methylated to an extent that depends on cell type. The degree of methylation in any case definitely increases during apoptosis. In the studied cell systems (human leukaemia, human prostate tumor, and rat thyroid transformed cells) among the low-molecular-mass HMG proteins, only HMGA1a was found to be methylated. A tryptic digestion map of HPLC-purified HMGA1a protein showed that methylation occurs at arginine 25 in the consensus G(24)R(25)G(26) that belongs to one of the DNA-binding AT-hooks of the protein. An increase of HMGA1a methylation could be related to heterochromatin and chromatin remodeling of apoptotic cells.     

The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing

This report describes a common method of obtaining template DNA from phagemids, phages and plasmids. The strategy is based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) for DNA precipitation. By avoiding phase separation, many manipulation steps are reduced. A time-saving modification to perform double-stranded DNA sequencing directly after alkaline-denaturation is also introduced. The protocols described here allow the researcher to obtain template DNA from a variety of initial sources, thus giving reproducible sequencing results when using T7 DNA polymerase.     

A fast method for high-quality genomic DNA extraction from whole human blood

A simple and fast protocol is described for the purification of genomic DNA from 0.3 ml of whole human blood. The recovery of DNA is quantitative and reproducible; the quality is such that it can be used for all relevant molecular biology techniques.     

Hepatitis-associated antigen in immunofluorescence I. Interfering factors

Synopsis By means of the indirect immunofluorescence test, Australia hepatitis-associated antigen (Au/SH) was studied in human liver cell suspensions obtained by needle biopsy. Fluorescence staining was performed with human anti-Au/SH immunoglobulins; controls with non-immune human and guinea-pig sera and with cellular substrates lacking Au/SH antigen (rat regenerating liver) were also carried out.Eight out of forty-one unselected patients showed specific fluorescence in their liver cells without any correlation with clinical diagnosis, presence of Au/SH antigen in the serum or of virus-like particles in the hepatocytes by ultrastructural examination. The positive cases were characterized by intranuclear fluorescent granules in, probably, nucleoli; this was confirmed by cytochemical investigations. However, the eight cases positive to anti-Au/SH antiserum showed a quite identical nucleolar fluorescence with non-immune human and guinea-pig sera. In addition, histochemical and ultrastructural examinations demonstrated, only in these positive cases, an increased nucleolar RNS-synthesis.The same results were obtained with cell suspensions of rat regenerating livers removed 20–24 hr after partial hepatectomy. These data strongly support that enhanced nucleolar RNA synthesis is responsible for non-specific positive fluorescence.Elution and digestion tests demonstrated that a heat-labile C'lq-like factor can interfere in the positivity of this fluorescence test.