Identification of subgroups of high-grade oligodendroglial tumors by comparative genomic hybridization.

In contrast to astrocytic tumors, approximately two thirds of anaplastic oligodendrogliomas are reported to be chemosensitive. Relatively little is known about the genetic aberrations in oligodendroglial tumors (OTs). In order to elucidate oligodendroglial oncogenesis and to find specific genetic aberrations that may have prognostic and therapeutic implications, we performed comparative genomic hybridization (CGH) to detect chromosomal copy number changes in 17 low-grade OTs (LG-OTs) and 12 high-grade OTs (HG-OTs) lacking a prominent astrocytic component. Loss of chromosome 1p (79%) and 19q (76%) were most frequently detected by CGH, all LG-OTs and 50% of the HG-OTs contained -1p (including 1p36-32), -19q (including 19q13.3), or both, and the rest of the HG-OTs showed +7, -10, or both. Since losses of 1p36-32 and 19q13.3 were mutually exclusive with +7 or -10, the HG-OTs could be divided in -1p/-19q and +7/-10 tumors. While the -1p/-19q tumors can be considered as pure anaplastic oligodendrogliomas, the +7/-10 tumors may rather be glioblastomas with prominent oligodendroglial differentiation. We conclude that CGH is a powerful tool to assist in the identification of 2 major subgroups of HG-OTs with prognostic and possibly therapeutic relevance.

Saponin pre-treatment in pre-embedding electron microscopic in situ hybridization for detection of specific RNA sequences in cultured cells: a methodological study.

We describe a method for detection of specific RNA targets in cultured cells at the electron microscopic (EM) level using pre-embedding in situ hybridization (ISH). The specimens were monitored by reflection-contrast microscopy (RCM) before processing for EM. A good balance between preservation of ultrastructure and intensity of hybridization signals was obtained by using mild aldehyde fixation followed by saponin permeabilization. Digoxigenin-labeled probes were used for detection of human elongation factor (HEF) mRNA in HeLa cells, immediate early (IE) mRNA in rat 9G cells, and 28S rRNA in both cell lines. The hybrids were detected immunocytochemically by the peroxidase/diaminobenzidine (DAB) method or by ultra-small gold with silver enhancement. Comparison of these methods favored the peroxidase/DAB system. The accessibility of RNA in the different cell compartments was dependent on the extent of cross-linking during primary fixation even after permeabilization with saponin. By using the most optimal ISH protocol and the peroxidase/DAB system, we detected 28S rRNA over all ribosomes in the cytoplasm but not in the nucleoli, and IE mRNA in a large spot with many smaller spots around it in the nucleoplasm as well as in speckles over the cytoplasm. The sensitivity of the method is such that HEF housekeeping gene transcripts were detected in the cytoplasm.

AgarCyto: a novel cell-processing method for multiple molecular diagnostic analyses of the uterine cervix.

In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer.

Monitoring morphology and signal during non-radioactive in situ hybridization procedures by reflection-contrast microscopy and transmission electron microscopy.

We analyzed the effects of steps in RNA in situ hybridization (ISH) procedures on morphology and hybridization signal with reflection-contrast microscopy (RCM) and transmission electron microscopy (TEM). In chessboard experiments, a range of fixatives containing formaldehyde, glutaraldehyde, or both, and various permeabilization protocols, including ethanol and pepsin treatment, were investigated. A transfected rat fibroblast cell line that harbors an inducible human cytomegalovirus immediate early (IE) transcription unit, and specific probes for 28S ribosomal RNA and IE messenger RNA were used for this purpose. Probes were labeled with digoxigenin and hybrids were detected with anti-digoxigenin F(ab)2 fragments conjugated to horseradish peroxidase, followed by diaminobenzidine/H2O2 reaction. Effects of fixation and pre-treatments on RNA detection efficiency and morphology were monitored by RCM on whole cells. After Epon embedding and ultra-thin cross-sectioning, the corresponding TEM images were obtained. With the pre-treatments analyzed, it appeared impossible to find an acceptable balance between ISH signals and preservation of ultrastructural morphology: when good signal-to-noise ratios are obtained, the ultrastructural morphology is already deteriorated. We discuss the parameters that influence the fragile balance between high RNA detection efficiency and good preservation of ultrastructure and the benefit of RCM monitoring in the development and procedures for pre-embedding electron microscopic ISH.

Genetic reflection of glioblastoma biopsy material in xenografts: characterization of 11 glioblastoma xenograft lines by comparative genomic hybridization.

OBJECT: Human tumors implanted as subcutaneous xenografts in nude mice are widely used for the study of tumor biology and therapy. Validation of these models requires knowledge of the genetic makeup of the xenografts. The aim of this study was to establish whether chromosomal imbalances in 11 xenograft lines derived from human glioblastomas multiforme (x-GBMs) are similar to those found in GBM biopsy samples. The authors also studied genetic stability during serial passaging of three xenograft lines. METHODS: Chromosomal imbalances in x-GBMs were detected using comparative genomic hybridization (CGH). The authors compared the CGH results in x-GBMs with those in the original GBMs (o-GBMs) that were used to establish three of the xenograft lines and with the GBM biopsy results reported in the literature (1-GBMs). In three xenograft lines two different passages were analyzed. CONCLUSIONS: The results show that the chromosomal imbalances in x-GBMs are similar to those in o-GBMs and 1-GBMs, indicating that the GBM xenograft lines used were valid models from a genetic point of view. The CGH analysis of two different passages of three xenograft lines indicates that x-GBMs (like 1-GBMs) show intratumoral genetic heterogeneity and do not acquire chromosomal imbalances as a result of serial passaging.

Noninvasive detection of fetal trisomy 21: systematic review and report of quality and outcomes of diagnostic accuracy studies performed between 1997 and 2012.

BACKGROUND: Research on noninvasive prenatal testing (NIPT) of fetal trisomy 21 is developing fast. Commercial tests have become available. To provide an up-to-date overview of NIPT of trisomy 21, an evaluation of the methodological quality and outcomes of diagnostic accuracy studies was made. METHODS: We undertook a systematic review of the literature published between 1997 and 2012 after searching PubMed, using MeSH terms 'RNA', 'DNA' and 'Down Syndrome' in combination with 'cell-free fetal (cff) RNA', 'cffDNA', 'trisomy 21' and 'noninvasive prenatal diagnosis' and searching reference lists of reported literature. From 79 abstracts, 16 studies were included as they evaluated the diagnostic accuracy of a molecular technique for NIPT of trisomy 21, and the test sensitivity and specificity were reported. Meta-analysis could not be performed due to the use of six different molecular techniques and different cutoff points. Diagnostic parameters were derived or calculated, and possible bias and applicability were evaluated utilizing the revised tool for Quality Assessment of Diagnostic Accuracy (QUADAS-2). RESULTS: Seven of the included studies were recently published in large cohort studies that examined massively parallel sequencing (MPS), with or without pre-selection of chromosomes, and reported sensitivities between 98.58% [95% confidence interval (CI) 95.9-99.5%] and 100% (95% CI 96-100%) and specificities between 97.95% (95% CI 94.1-99.3%) and 100% (95% CI 99.1-100%). None of these seven large studies had an overall low risk of bias and low concerns regarding applicability. MPS with or without pre-selection of chromosomes exhibits an excellent negative predictive value (100%) in conditions with disease odds from 1:1500 to 1:200. However, positive predictive values were lower, even in high-risk pregnancies (19.7-100%). The other nine cohort studies were too small to give precise estimates (number of trisomy 21 cases: ≤25) and were not included in the discussion. CONCLUSIONS: NIPT of trisomy 21 by MPS with or without pre-selection of chromosomes is promising and likely to replace the prenatal serum screening test that is currently combined with nuchal translucency measurement in the first trimester of pregnancy. Before NIPT can be introduced as a screening test in a social insurance health-care system, more evidence is needed from large prospective diagnostic accuracy studies in first trimester pregnancies. Moreover, we believe further assessment, of whether NIPT can be provided in a cost-effective, timely and equitable manner for every pregnant woman, is required.

Congenital hydrocephalus in clinical practice: a genetic diagnostic approach.

Congenital hydrocephalus is a common and often disabling disorder. The etiology is very heterogeneous. Little is known about the genetic causes of congenital hydrocephalus. A retrospective survey was performed including patients with primary congenital hydrocephalus referred to the Department of Clinical Genetics between 1985 and 2010 by perinatologists, (child) neurologists or pediatricians. Patients with hydrocephalus secondary to other pathology were excluded from this survey. We classified patients with primary congenital hydrocephalus into two main groups: non-syndromic hydrocephalus (NSH) and syndromic hydrocephalus (SH). Seventy-five individuals met the inclusion criteria, comprising 36% (27/75) NSH and 64% (48/75) SH. In 11% (8/75) hydrocephalus was familial. The cause of hydrocephalus was unknown in 81% (61/75), including all patients with NSH. The male-female ratio in this subgroup was 2.6:1, indicating an X-linked factor other than the L1CAM gene. In the group of SH patients, 29% (14/48) had a known cause of hydrocephalus including chromosomal abnormalities, L1 syndrome, Marden-Walker syndrome, Walker-Warburg syndrome and hemifacial microsomia. We performed this survey in order to evaluate current knowledge on the genetic etiology of primary congenital hydrocephalus and to identify new candidate genes or regulatory pathways for congenital hydrocephalus. Recommendations were made concerning the evaluation and genetic workup of patients with primary congenital hydrocephalus. We conclude that further molecular and functional analysis is needed to identify new genetic forms of congenital hydrocephalus.

Evaluation of pepsin treatment for electron microscopic RNA in situ hybridization on ultra-thin cryosections of cultured cells.

The in situ hybridization (ISH) technique, as applied to electron microscopic detection of RNAs, was evaluated for ultra-thin cryosections of cultured rat fibroblasts (rat 9G). Experimental variables to balance penetration of detection reagents and preservation of ultrastructural morphology included various strengths of aldehyde fixation and pepsin treatment. We performed ISH for 28S ribosomal RNA (rRNA) followed by ultra-small colloidal gold immunocytochemistry and silver enhancement. An acceptable balance for 28S rRNA ISH detection was obtained using mild cross-linking fixation followed by treatment with a relative high concentration of pepsin for a short time. The ISH method presented in this study was compatible with immunocytochemical detection of protein as demonstrated by double-labeling experiments.

Absence of karyotype abnormalities in patients with familial urothelial cell carcinoma.

OBJECTIVES: In a previous pilot study, a constitutional balanced translocation t(5;20)(p15;q11) was identified in a family with urothelial cell carcinoma (UCC). The purpose of this study was to find (additional) constitutional chromosomal abnormalities in selected families to obtain an indication for genome location(s) of UCC susceptibility gene(s). METHODS: UCC families were selected through an ongoing study on familial clustering of UCC, the largest study on this subject ever performed. This study included 1193 new patients with UCC of the bladder, ureter, and renal pelvis, identified from the population-based cancer registries of the Dutch Comprehensive Cancer Centers East and South. Information on demographic factors, smoking habits, and family history of UCC was collected by postal questionnaires. UCC in the families was verified with pathology reports. Thirty families were selected in which 2 or 3 individuals were affected, preferably diagnosed at a relatively young age. Blood samples were obtained from all probands, and routine cytogenetic analysis was performed on 23 male and 7 female UCC patients. Subsequent spectral karyotyping was performed in 4 patients from families, which were most suggestive for an inherited etiology. RESULTS: No aberrant chromosomal features were found by either classical or spectral karyotype analyses. CONCLUSIONS: It is conceivable that genetic germline abnormalities do exist in the patients in our study but are below the detection limit of the explorative methods used in this study.

Imaging of RNA in situ hybridization by atomic force microscopy.

In this study we investigated the possibility of imaging internal cellular molecules after cytochemical detection with atomic force microscopy (AFM). To this end, rat 9G and HeLa cells were hybridized with haptenized probes for 28S ribosomal RNA, human elongation factor mRNA and cytomegalovirus immediate early antigen mRNA. The haptenized hybrids were subsequently detected with a peroxidase-labelled antibody and visualized with 3.3'-diaminobenzidine (DAB). The influence of various scanning conditions on cell morphology and visibility of the signal was investigated. In order to determine the influence of ethanol dehydration on cellular structure and visibility of the DAB precipitate, cells were kept in phosphate-buffered saline (PBS) and scanned under fluid after DAB development or dehydrated and subsequently scanned dry or submerged in PBS. Direct information on the increase in height of cellular structures because of internally precipitated DAB and the height of mock-hybridized cells was available. Results show that internal DAB precipitate can be detected by AFM, with the highest sensitivity in the case of dry cells. Although a relatively large amount of DAB had to be precipitated inside the cell before it was visible by AFM, the resolution of AFM for imaging of RNA--in situ hybridization signals was slightly better than that of conventional optical microscopy. Furthermore, it is concluded that dehydration of the cells has irreversible effects on cellular structure. Therefore, scanning under fluid of previously dehydrated samples cannot be considered as a good representation of the situation before dehydration.

Spectral imaging of multi-color chromogenic dyes in pathological specimens.

We have investigated the use of spectral imaging for multi-color analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier-transform spectroscopy and digital imaging. A pixel-by-pixel spectrum-based color classification is presented of single-, double-, and triple-color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki-67 and TP53 in paraffin-embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright-field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference library. We conclude that spectral imaging of multi-color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell- and tissue specimens.