Rapid generation of chromosome-specific alphoid DNA probes using the polymerase chain reaction

Non-isotopic in situ hybridization of chromosome-specific alphoid DNA probes has become a potent tool in the study of numerical aberrations of specific human chromosomes at all stages of the cell cycle. In this paper, we describe approaches for the rapid generation of such probes using the polymerase chain reaction (PCR), and demonstrate their chromosome specificity by fluorescence in situ hybridization to normal human metaphase spreads and interphase nuclei. Oligonucleotide primers for conserved regions of the alpha satellite monomer were used to generate chromosome-specific DNA probes from somatic hybrid cells containing various human chromosomes, and from DNA libraries from sorted human chromosomes. Oligonucleotide primers for chromosome-specific regions of the alpha satellite monomer were used to generate specific DNA probes for the pericentromeric heterochromatin of human chromosomes 1, 6, 7, 17 and X directly from human genomic DNA

Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84

The localization of chromosome 18 in human interphase nuclei is demonstrated by use of radioactive and nonradioactive in situ hybridization techniques with a DNA clone designated L1.84. This clone represents a distinct subpopulation of the repetitive human alphoid DNA family, located in the centric region of chromosome 18. Under stringent hybridization conditions hybridization of L1.84 is restricted to chromosome 18 and reflects the number of these chromosomes present in the nuclei, namely, two in normal diploid human cells and three in nuclei from cells with trisomy 18. Under conditions of low stringency, cross-hybridization with other subpopulations of the alphoid DNA family occurs in the centromeric regions of the whole chromosome complement, and numerous hybridization sites are detected over interphase nuclei. Detection of chromosome-specific target DNAs by non-radioactive in situ hybridization with appropriate DNA probes cloned from individual chromosomal subregions presents a rapid means of identifying directly numerical or even structural chromosome aberrations in the interphase nucleus. Present limitations and future applications of interphase cytogenetics are discussed

Proliferation and aneusomy predict survival of young patients with astrocytoma grade II

The clinical course of astrocytoma grade II (AII) is highly variable and not reflected by histological characteristics. As one of the best prognostic factors, higher age identifies rapid progressive A II. For patients over 35 years of age, an aggressive treatment is normally propagated. For patients under 35 years, there is no clear guidance for treatment choices, and therefore also the necessity of histopathological diagnosis is often questioned. We studied the additional prognostic value of the proliferation index and the detection of genetic aberrations for patients with A II. The tumour samples were obtained by stereotactic biopsy or tumour resection and divided into two age groups, that is 18–34 years (n=19) and 35 years (n=28). Factors tested included the proliferation (Ki-67) index, and numerical aberrations for chromosomes 1, 7, and 10, as detected by in situ hybridisation (ISH). The results show that age is a prognostic indicator when studied in the total patient group, with patients above 35 years showing a relatively poor prognosis. Increased proliferation index in the presence of aneusomy appears to identify a subgroup of patients with poor prognosis more accurately than predicted by proliferation index alone. We conclude that histologically classified cases of A II comprise a heterogeneous group of tumours with different biological and genetic constitution, which exhibit a highly variable clinical course. Immunostaining for Ki-67 in combination with the detection of aneusomy by ISH allows the identification of a subgroup of patients with rapidly progressive A II. This is an extra argument not to defer stereotactic biopsy in young patients with radiological suspicion of A II

Distribution Pattern and Marker Profile Show Two Subpopulations of Reserve Cells in the Endocervical Canal

A previous immunophenotyping Study ill the fetal uterine cervix provided evidence for the existence of 2 subpopulations of reserve cells, one giving rise to glandular epithelium and the other to squamous epithelium (5). Ill this Study, we investigated whether the adult Uterine cervix also harbors different populations Of reserve cells oil the basis of their marker profile and distribution pattern. Sagittal sections from 10 normal uteri, comprising the region from ectocervix to lower Uterine cavity, were histologically examined and immunostained for p63, bcl-2 and cytokeratins (CKs) 5, 7, 8, and 17. The endocervical canal consists of three regions, that is, it part lined with squamous epithelium, a part lined with endocervical cells and a part lined With tubal type epithelial cells. Histologically, We found reserve cells ill all 10 investigated cervices, with,in abundancy ill the area beneath the endocervical columnar epithelium close to the squamo-columnar junction, and high ill the endocervical canal where the invaginations consist of tubal type epithelium. In between, all area lined with endocervical columnar cells without reserve cells was identified. No reserve cells were detected in the endometrial epithelium. We defined the end of the endocervix as the point where the Surface of the cervical canal and the invaginations are completely lined with tubal type epithelium. From this point, reserve cells were no longer found. Reserve cells show strong expression for p63, CKs 5 and 7. and moderate expression for bcl-2. CK 17 is strongly expressed in the reserve cells at the squamo-columnar junction and to a lesser extent in the reserve cells close to the endometrium. Endocervical columnar cells usually express CKs 7 and 8 and sporadically also p63 and CK5. CK 17 was only found in endocervical cells in the vicinity of CK 17-positive subcolumnar reserve cells. Tubal-type epithelium was present in all samples and contained bcl-2, along with CKs 5, 7, and 8. As a result, bcl-2 and CK5 expression distinguishes tubal epithelium from endocervical columnar cells. We conclude that reserve cells are present in all investigated cervices along the entire cervical canal. The concentration Of subglandular reserve cells is highest close to the squamo-columnar junction and in the upper third of the cervix. The marker profile of reserve cells is the same in all parts of the cervix, except for CK 17, which shows a decreasing gradient from distal to proximal, indicating a subpopulation of distal reserve cells as progenitor for squamous and columnar epithelium. and proximal reserve cells that can serve as progenitor cells for columnar epithelium