Genotypic characterization of prostatic carcinomas: a combined cytogenetic, flow cytometry, and in situ DNA hybridization study.

Cytogenetic studies were performed on 36 biopsies obtained from 26 primary prostatic adenocarcinomas. Following histopathological characterization of control sections, the biopsies were investigated using metaphase cytogenetics, DNA flow cytometry, and fluorescence in situ DNA hybridization. In 12 specimens, no carcinoma was found in control sections by histopathological means. In 24 carcinoma biopsies clonal aberrations were detected in 15 specimens. Tetraploidy as sole aberration was detected in five specimens. Loss of the Y chromosome was seen in eight samples. Only one tumor revealed structural abnormalities. Eight samples were found to be normal (46,XY). Remarkably, nonclonal chromosome aberrations, particularly marked chromosome loss, were frequently detected in prostatic carcinomas and premalignant lesions (prostatic intraepithelial neoplasia). In the series of biopsies investigated by means of cytogenetics and flow cytometry, biopsies with aneuploid DNA content were found to be cytogenetically normal. Conversely, the cytogenetically aberrant clones were found to be of diploid DNA content. Evidence of focal intratumoral heterogeneity was revealed by cytogenetics, flow cytometry, and in situ hybridization.

Comparative genomic hybridization as part of a new diagnostic strategy in childhood hyperdiploid acute lymphoblastic leukemia.

The detailed definition of karyotype changes associated with hyperdiploid acute lymphoblastic leukemia (ALL) is a precondition for their exploitation in minimal residual disease studies with fluorescence in situ hybridization analysis (FISH). In addition, certain karyotype patterns may have different prognostic implications. We have therefore used comparative genomic hybridization (CGH) to analyze the quantitative karyotype abnormalities in 14 cases of hyperdiploid ALL and correlated the results with those obtained by flow cytometry and conventional cytogenetic analyses. Despite an overall good agreement between the karyotypes obtained by classical banding techniques and CGH, we came across at least one karyotype discrepancy per case. Clarification of the discordant findings with fluorescence in situ hybridization (FISH) showed that all stem lines had been correctly defined by CGH. In eight cases, however, cytogenetic analyses revealed structural abnormalities that were undetectable by CGH. The other discrepancies were mainly due to a cytogenetic misinterpretation of similar sized and shaped chromosomes. Based on these findings we present a new diagnostic strategy for childhood ALL that includes flow cytometry and classical cytogenetics as well as CGH for the analysis of aneuploid cases and FISH to resolve the unavoidable discrepancies.

Induction of apoptosis by an inhibitor of cAMP-specific PDE in malignant murine carcinoma cells overexpressing PDE activity in comparison to their nonmalignant counterparts.

In order to study potential changes in phosphodiesterase (PDE) activity associated with malignant transformation, normal primary keratinocytes and cells corresponding to different stages of epidermal tumor development in mouse skin were analyzed with respect to their 3',5'-cyclic adenosine monophosphate (cAMP) hydrolyzing activity. Expression of cAMP-specific PDE-4, intracellular cAMP content, and the sensitivity to the growth inhibitory effect of the PDE-4-specific inhibitor 7-benzylamino-6-chloro-2 piperazino-4-pyrrolidino-pteridine (DC-TA-46) were studied in the two papilloma cell lines, MSCP6 and 308, and in the highly malignant carcinoma cell line CarB. No significant difference in soluble PDE activity and in intracellular cAMP was found in the two papilloma cell lines when compared to primary keratinocytes. In contrast, the spindle-cell carcinoma cell line CarB exhibited significantly higher PDE activity, concomitant with the lowest cAMP level. In all cell lines and also in the primary keratinocytes, rolipram-sensitive PDE-4 activity accounted for the major cAMP-hydrolyzing activity. In primary keratinocytes and in MSCP6 cells, the PDE-4 inhibitor DC-TA-46 induced at best marginal growth inhibition, whereas cell growth of 308 cells was markedly affected at concentrations > 2 microM. The carcinoma cell line CarB showed the highest sensitivity to DC-TA-46 (IC50 = 0.8 +/- 0.3 microM). Treatment of CarB cells with DC-TA-46 strongly inhibits intracellular PDE activity, resulting in a marked and long-lasting rise of cAMP. After 24 h of treatment, arrest in the G0/G1 phase of the cell cycle is induced. Treatment with concentrations > 2 microM of this highly effective PDE inhibitor results in induction of apoptotic cell death, as detected by fluorescence microscopy, flow cytometry, and ELISA-based determination of fragmented DNA in intact cells.

DNA aneuploidy in prostatic adenocarcinoma: a frequent event as shown by fluorescence in situ DNA hybridization.

Fluorescence in situ hybridization (FISH) using specific DNA probes for chromosomes 1, 7, 10, and Y was performed on 53 prostatic tissue samples obtained from 33 radical prostatectomy specimens and two benign control specimens. The 53 samples from carcinomatous prostates included 33 cancerous and 20 noncancerous samples. Additionally, four metastatic lymph node specimens were examined. Clonal chromosome abnormalities were observed in 78% of the tumors studied. They were detected in a higher proportion in stage pT2 and pT3 tumors (86% and 88%, respectively) compared with stage pT1 tumors (25%). No stage pT4 tumor was analyzed. There was evidence of remarkable focal intratumoral heterogeneity documented by the study of two samples from the same tumor in three of six cases. Comparing FISH determined ploidy patterns with DNA flow cytometry (FCM) in 22 samples, FISH showed aneuploidy whereas FCM showed none.

Microsatellite instability in sporadic carcinomas of the proximal colon: association with diploid DNA content, negative protein expression of p53, and distinct histomorphologic features.

BACKGROUND: Microsatellite instability (MIN) seems to characterize a particular subset of sporadic colorectal adenocarcinomas with the studies indicating a better clinical outcome for patients with MIN-positive tumors than for those with MIN-negative ones. The goal of this study was to further clarify whether a genotype-specific histomorphology of the right-sided colonic carcinomas can be identified. METHODS: MIN status, DNA content, and p53 protein expression were evaluated in cryoconserved specimens from 20 adenocarcinomas of the proximal colon and correlated to stage, grade, and other histomorphologic features. The study was restricted to tumors of the proximal colon because approximately 90% of all MIN-positive tumors were found in the proximal colon, and differences between right- and left-sided tumors cannot be excluded a priori. RESULTS: By using four microsatellite markers, instability was detected in 35% of the tumors analyzed. The clinicopathologic features in the MIN-positive tumors were found to differ markedly from the MIN-negative tumors in their poorly differentiated histologic pattern, extracellular mucin production, and favorable lymph node and distant metastatic behavior. A marked association was found between MIN positivity and DNA diploid status, as well as negative p53 immunostaining. CONCLUSIONS: The MIN-positive colonic carcinomas were characterized by distinct histomorphologic features that are recognizable at routine diagnostic evaluation. Poorly differentiated adenocarcinomas of the proximal colon, with only a few lymph nodes and no distant metastases at presentation, and lack of p53 accumulation are highly suggestive of being MIN positive. These tumors should be discriminated from the other poorly differentiated carcinomas, because they seem to be associated with an improved prognosis compared with the tumors without microsatellite instability.

Rearrangement of chromosome 1 is a frequent finding in endometrial carcinoma. An in situ hybridization study in nine endometrial carcinomas.

Nine endometrial carcinomas were examined for numerical aberrations of the chromosomes 1,7, and X by fluorescence in situ hybridization using highly repetitive chromosome-specific probes. In addition, a combination of a centromeric and a telomeric chromosome 1 probe was applied to detect structural chromosome 1 aberrations. Chromosome aberrations were found in six tumors. In four of these, an imbalance between 1q12 and 1p36 was detected, indicating the presence of an extra 1p- chromosome. In regard to the chromosomes 7 and X, monosomies and trisomies were found. Intratumoral genetic heterogeneity in endometrial carcinomas was detectable by FISH and flow cytometry. In conclusion, our findings confirm that chromosome 1 is frequently involved in structural chromosome changes, indicating chromosome 1 to be of importance in the evolution of endometrial carcinoma.

Chromosomal homeologies between human, harbor seal (Phoca vitulina) and the putative ancestral carnivore karyotype revealed by Zoo-FISH.

We report on the construction of the first comparative Zoo-FISH map of a marine mammal. Zoo-FISH with DNA probes from a human chromosome-specific library to metaphase spreads of the harbor seal (Phoca vitulina) disclosed 31 conserved syntenic segments covering the complete autosomal complement and the X chromosome. Comparison with Zoo-FISH maps of other species reveals that the harbor seal shares a high degree of karyotypic homeology with the human complement and an even higher degree with the conordinal cat complement. These findings suggest that pinniped, felid and human karyotypes have maintained conserved complements. Based on data of Zoo-FISH and comparative cytogenetics, a Zoo-FISH map of the ancestral carnivore karyotype (Z-CAR) is proposed. Flow cytometry revealed that the DNA value of the harbor seal genome is 79% that of the human genome.

Comparative genomic in situ hybridization discloses chromosomal copy number changes in a transplanted brain tumor line of the rat (Rattus norvegicus).

We investigated chromosomal copy number changes in ethylnitrosourea-induced and serially transplanted gliomas of the rat by flow cytometry and Comparative Genomic in situ Hybridization (CGH). CGH analysis of a primary and four transplanted tumors revealed several genomic aberrations, including whole chromosome and subchromosomal gains and losses. Gains involved rat Chromosomes (RNO) 2, 3, 4, 5, 7, 9, 11, 12, 13, and Y, whereas losses affected RNO5, 13, 20, and Y. The primary tumor exhibited gain of RNO2q31qter and gain of RNO4. While gain of RNO2 was seen in nearly all investigated passages, gain of RNO4 was apparent in the primary tumor and in passage 2 and 5 tumors. Chromosomal alterations detected as single events were restricted to the transplanted tumors and included gain of RNO3q11, 3q41qter, 5q36, 7q34qter, 9q37, 11q, and Y, and loss of RNO5, 13, and 20q. Flow cytometry disclosed different aneuploid cell clones in the tumors investigated. The results are discussed in analogy to findings in human glial tumors.

Selection toward diploid cells in prostatic carcinoma derived cell cultures.

For elucidation of the growth-regulatory mechanisms in prostatic carcinoma, in vitro investigations on prostatic cell cultures are required. However, one major problem of cell culturing is the selection of particular cell types such that the cell lines representing only some of the features as compared with the tumor of origin. We studied the chromosomal composition of 20 prostatic tissue-derived cell cultures and 12 original (fresh) tissue specimens that were obtained from 13 patients with prostatic adenocarcinoma. Using fluorescence in situ DNA hybridization (FISH), evident clonal abnormalities were detected in 78% of the fresh cancer samples and in 47% of the cultured cancer samples. Of the seven cases revealing clonal abnormalities in the fresh cancer specimen, aneuploidy was detected in only two samples after cell culturing at the earliest passage studied. The aneuploid cell populations in the cultured samples were all lost during progressive subcultivation (after passage 4). Interestingly, by performing FISH on cytogenetic preparations aneuploidy was confined to the interphases, with the metaphases being found to be diploid. This finding indicates that the aneuploid cells have a proliferation disadvantage in cell culture resulting in an overgrowth of diploid cells.

Lack of DNA synthesis among CD34+ cells in cord blood and in cytokine-mobilized blood.

Flow cytometric DNA analysis was performed in combination with three-colour immunological staining of cell surface antigens on density-separated mononuclear cells (MNC) obtained from peripheral blood (PB) before, during and after cytokine stimulation of healthy adults. The aim of the study was to determine the cell-cycling status of haemopoietic progenitor cells mobilized into the blood of healthy volunteers during a 5 d treatment period with 5/micrograms per kg body weight of either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-simulating factor (GM-CSF). Despite considerably increasing numbers of CD34+ PB MNC, the latter were not found to be in S/G2M phase, whereas, among the CD34- MNC, the proportion of cells in S/G2M phase increased from < 0.1% to 0.75 +/- 0.4% (GM-CSF) and to 1.34 +/- 0.75% (G-CSF) and dropped again after discontinuation of the cytokine stimulation. These cells expressed CD33 but were negative for CD45RA, CD3, CD19 and CD14 and were thus considered granulopoietic cells. Analogous results were obtained from analyses of cord blood (CB). In contrast, CD34+ cells from bone marrow (BM) were partially (between 9% and 15%) found to be in S/G2M phase. The non-cycling status of PB and progenitor cells was confirmed by the analysis of CD34+ cells enriched from the two cells sources. However, in vitro stimulation of these progenitor cells using IL3, GM-CSF, erythropoietin and steel factor (SF) revealed that, after 48 h in suspension culture, up to 30% of the CD34+ cells were in S/G2m phase. The fact that cycling CD34+ cells are only detectable in BM but not in PB or CB may suggest different adhesive properties of migrating/mobilized 'stem cells' which may require the BM micro-environment for adequate proliferation in vivo.