Redefining the risks of prenatally ascertained supernumerary marker chromosomes: a collaborative study.

BACKGROUND: A marker chromosome is defined as a structurally abnormal chromosome that cannot be identified by routine cytogenetics. The risk for phenotypic abnormalities associated with a marker chromosome depends on several factors, including inheritance, mode of ascertainment, chromosomal origin, and the morphology, content, and structure of the marker. METHODS: to understand the karyotype-phenotype relationship of prenatally ascertained supernumerary de novo marker chromosomes, we combined data from prenatal cases obtained from 12 laboratories with those from studies in the literature. We were able to obtain cytogenetic and phenotypic data from 108 prenatally ascertained supernumerary de novo marker chromosomes to refine the phenotypic risk associated with these markers. Because of the growing number of cases and because more techniques are available to delineate marker morphology, we have been able to group risk estimates into subcategories, such as by marker type and whether there are ultrasound abnormalities. RESULTS: If a de novo supernumerary marker chromosome is found prenatally, our data suggest there is a 26% risk for phenotypic abnormality when there is no other information defining the marker (such as chromosomal origin or information about the existing phenotype). However, if high resolution ultrasound studies are normal, this risk reduces to 18%. CONCLUSIONS: Our findings strongly support the value of additional genetic studies for more precisely defining the risk in individual cases involving marker chromosomes.

Human cell lines as an in vitro/in vivo model for prostate carcinogenesis and progression.

BACKGROUND: The study of prostate carcinogenesis and tumor progression is made difficult by the lack of appropriate in vitro and in vivo models. High prevalence of prostatic intra-epithelial neoplasia and latent prostatic carcinoma, representing multiple steps in carcinogenesis to invasive carcinoma, are relevant targets for cancer prevention. From the RWPE-1, immortalized, non-tumorigenic, human prostate epithelial cell line, we have derived four tumorigenic cell lines with progressive malignant characteristics. METHODS: Cell lines were derived by exposure of RWPE-1 to N-methyl-N-nitrosourea (MNU), selected and cloned in vivo and in vitro, and characterized by prostatic epithelial and differentiation markers, karyotype analysis, anchorage-independent growth, invasiveness, tumorigenicity, and pathology of the derived tumors. RESULTS: Cytokeratins 8 and 18, androgen receptor, and prostate-specific antigen expression in response to androgen, confirm prostatic epithelial origin. RWPE-1 cells do not grow in agar and are not tumorigenic in mice, but the growth, tumorigenicity, and tumor pathology of the MNU cell lines correlate with their invasive ability. The WPE1-NA22 (least malignant) form small, well-differentiated, and WPE1-NB26 cells (most malignant) form large, poorly differentiated, invasive tumors. Overall, loss of heterozygosity for chromosomes 7q, 13q, 18q, and 22, and gain of 5, 9q, 11q, and 20, was observed. The MNU cell lines, in order of increasing malignancy are; WPE1-NA22, WPE1-NB14, WPE1-NB11, and WPE1-NB26. CONCLUSIONS: This family of cell lines with a common lineage represents a unique and relevant model which mimics stages in prostatic intra-epithelial neoplasia (PIN) and progression to invasive cancer, and can be used to study carcinogenesis, progression, intervention, and chemoprevention.

A multicenter investigation with D-FISH BCR/ABL1 probes.

Twenty-eight laboratories evaluated a new fluorescence in situ hybridization (FISH) strategy for chronic myeloid leukemia. In a three-part study, bcr/abl1 D-FISH probes were used to study bone marrow specimens. First, laboratories familiarized themselves with the strategy by applying it to known normal and abnormal specimens. Then, collectively the laboratories studied 20 normal and 20 abnormal specimens blindly and measured workload. Finally, each laboratory and two experts studied six serial dilutions with 98-0% abnormal nuclei. Using the reported normal cutoff of < 1% abnormal nuclei, participants reported no false-negative cases and 15 false-positive cases (1-6.6% abnormal nuclei). Results provided by participants for serial dilutions approximated the expected percentages of abnormal nuclei, but those from the experts exhibited greater precision. The clinical sensitivity, precision, nomenclature, workload, recommendations for training, and quality assurance in methods using D-FISH in clinical practice are discussed.

A human prostatic stromal myofibroblast cell line WPMY-1: a model for stromal-epithelial interactions in prostatic neoplasia.

Here we report the characterization of an SV40 large-T antigen-immortalized stromal cell line, WPMY-1, derived from the same prostate as our previously described epithelial cell lines. The WPMY-1 cells were determined to be myofibroblasts on the basis of co-expression of smooth muscle alpha-actin and vimentin. They also show positive staining for androgen receptor, large-T antigen, and positive but heterogeneous staining for p53 and pRb. Their growth is stimulated by the synthetic androgen mibolerone to 145% of control (100%). Platelet-derived growth factor BB, epidermal growth factor and basic fibroblast growth factor, at 10 ng/ml, stimulated growth to 138, 143 and 146% of control, respectively. Transforming growth factor-beta, at 10 ng/ml, inhibited serum-induced growth to 65% of control in the presence of 1% serum, and bFGF-induced growth to 30% of control. A serum-free medium was developed for optimal growth of WPMY-1 cells. They show anchorage-independent growth in soft agar. Studies on paracrine interactions show that myofibroblast-conditioned medium causes a marked inhibition of growth in WPE1-10 cells, while conditioned medium from WPE1-10 prostatic epithelial cells caused only a small increase in the growth of WPMY-1 cells. WPMY-1 cells secrete very low levels of MMP-9 but high levels of MMP-2, markedly higher than the epithelial cells. These epithelial and myofibroblast cell lines, derived from the same prostate, provide novel and useful models for studies on paracrine stromal-epithelial interactions in carcinogenesis, tumor progression, prevention and treatment of prostate cancer and benign prostatic hyperplasia.

Toward quality assurance for metaphase FISH: a multicenter experience.

Although fluorescent in situ hybridization (FISH) is rapidly becoming a part of clinical cytogenetics, no organization sponsors multicenter determinations of the efficacy of probes. We report on 23 laboratories that volunteered to provide slides and to use a probe for small nuclear ribonucleoprotein polypeptide N (SNRPN) and a control locus. Experiences with FISH for these laboratories during 1994 ranged from 0 to 645 utilizations (median = 84) involving blood, amniotic fluid, and bone marrow. In an initial study of hybridization efficiency, the median percentage of metaphases from normal individuals showing two SNRPN and two control signals for slides prepared at each site was 97.0 (range = 74-100); for slides prepared by a central laboratory, it was 97.8 (range = 81.6-100). In a subsequent blind study, each laboratory attempted to score 5 metaphases from each of 23 specimens [8 with del(15)(q11.2-->q12) and 15 with normal #15 chromosomes]. Of 529 challenges, the correct SNRPN pattern was found in 5 of 5 metaphases in 457 (86%) and in 4 of 5 in 33 (6%). Ambiguous, incomplete, or no results were reported for 32 (6%) challenges. Seven (1%) diagnostic errors were made, including 6 false positives and 1 false negative: 1 laboratory made 3 errors, 1 made 2, and 2 made 1 each. Most errors and inconsistencies seemed due to inexperience with FISH. The working time to process and analyze slides singly averaged 49.5 min; slides processed in batches of 4 and analyzed singly required 36.9 min. We conclude that proficiency testing for FISH by using an extensive array of challenges is possible and that multiple centers can collaborate to test probes and to evaluate costs.

Toward quality assurance for metaphase FISH: a multi-center experience.

Although fluorescent in situ hybridization (FISH) is rapidly becoming a part of clinical cytogenetics, no organization sponsors multi-center determinations of the efficacy of probes. We report on 23 laboratories that volunteered to provide slides and to use a probe for SNRPN and a control locus. Experiences with FISH for these laboratories during 1994 ranged from 0 to 645 utilizations (median = 84) involving blood, amniotic fluid and bone marrow. In an initial study of hybridization efficiency, the median percentage of metaphases from normal individuals showing two SNRPN and 2 control signals for slides prepared at each site was 97.0 (range = 74-100); for slides prepared by a central laboratory, it was 97.8 (range = 81.6-100). In a subsequent blind study, each laboratory attempted to score 5 metaphases from each of 23 specimens [8 with del(15) (q11.2-->q12) and 15 with normal 15 chromosomes]. Of 529 challenges, the correct SNRPN pattern was found in 5 of 5 metaphases in 457 (86%) and in 4 of 5 in 33 (6%). Ambiguous, incomplete or no results were reported for 32 (6%) challenges. Seven (1%) diagnostic errors were made including 6 false positives and 1 false negative: 1 laboratory made 3 errors, 1 made 2, and 2 made 1 each. Most errors and inconsistencies seemed due to inexperience with FISH. The working time to process and analyze slides singly averaged 49.5 minutes; slides processed in batches of 4 and analyzed singly required 36.9 minutes. We conclude that proficiency testing for FISH using an extensive array of challenges is possible and that multiple centers can collaborate to test probes and to evaluate costs.

Non-mosaic tetrasomy 9p in a liveborn infant with multiple congenital anomalies: case report and comparison with trisomy 9p.

Tetrasomy of the short(p) arm of chromosome 9 has been reported in few cases. Most of these children present with microbrachycephaly, wide forehead, hypertelorism, lowset, malformed ears, beaked noses, and micrognathia. Additional anomalies include short neck, congenital heart disease, genital abnormalities, multiple limb defects, hypotonia, and early death.

Clonal chromosomal aberrations in a leiomyosarcoma of the sinonasal tract.

We report the first cytogenetic analysis of a leiomyosarcoma of the sinonasal tract, a rare neoplasm. Karyotypic analysis showed near-triploid and near-tetraploid modal chromosome numbers with extensive structural and numerical aberrations. Three consistent structural changes, including i(6p), der(10)ins(10;1)(q26;q23q44), and der(12)t(1;12)(q11;q24) were observed in most cells. A der(11)t(11;?)(p15;?) was observed in 14 of 20 cells. Clonal structural rearrangements, including i(1q), del(2)(q37), der(3)t(3;?)(p25;?), del(4)(q31), del(7)(q32), der(12)t(12;?)(p12;?), der(15), del(21)(q22), and der(X) were each observed in a few cells. Numerical changes, including trisomies for chromosomes 2-5, 7, 9, 11, 15, 17, 18, and 20 and monosomies 10 and 12 were observed. Comparison of our findings to those of leiomyosarcomas at different sites showed trisomies 7 and 20 and rearrangements of 11p12-p15 and 21q22.

Two new human cholangiocarcinoma cell lines and their cytogenetics and responses to growth factors, hormones, cytokines or immunologic effector cells.

Two new human cholangiocarcinoma (CC) cell lines (CC-SW-I and CC-LP-I) were established and maintained in culture for 2 years. Histologically, both original liver tumors were adenocarcinomas, and the cell lines exhibited morphologic features of moderately differentiated adenocarcinoma. Immunohistochemistry showed that both cell lines were strongly positive for cytokeratin AEI but negative for carbohydrate tumor-associated antigen, CA19-9. Ultrastructural analysis of both cell lines showed the presence of tight junctional complexes and focally formed microvilli. Both CC cell lines were tumorigenic in nude mice. Cytogenetic analysis showed that both cell lines expressed highly aneuploid karyotypes with numerous structural and numerical deviations. CC-SW-I was hypodiploid with numerous chromosome losses and structural rearrangements, while CC-LP-I was hyperdiploid and displayed multiple additional chromosomes. Doubling times for the CC-SW-I and CC-LP-I cell lines in the presence of 15% fetal bovine serum were 72 hr and 180 hr, respectively. Growth of the CC-SW-I cell line was significantly stimulated in the presence of insulin, while that of the CC-LP-I cell line was significantly augmented by epidermal growth factor (EGF). In contrast, dexamethasone strongly inhibited proliferation of both cell lines in a dose-dependent manner. Among various recombinant cytokines examined for effects on growth or surface antigen expression on CC cell lines, only interleukin I-beta (ILI-beta) strongly inhibited growth of the CC-LP-I cell line, while interferons (IFNs) or tumor necrosis factor-alpha (TNF-alpha) were mildly inhibitory. Both tumor cell lines were resistant to natural killer (NK) cells but sensitive to lymphokine-activated killer (LAK) cells. Preincubation of tumor cells with IFN-gamma, IFN-alpha or TNF-alpha significantly decreased the susceptibility of each tumor cell line to lysis by LAK cells, and the change in sensitivity did not correlate with the expression of HLA antigens or intercellular adhesion molecule-I (ICAM-I) on the surface of tumor cells. These 2 CC cell lines are expected to provide valuable information about cell biology of human CC.

Confirmation of true mosaic trisomy 20 in a phenotypically normal liveborn male.

A new case of prenatally detected mosaic trisomy 20 (79% trisomy 20 cells in amniocyte cultures) that was confirmed in newborn tissue is presented. A healthy male infant was delivered at term, with no dysmorphology or apparent malformations; this baby is developing normally. Twenty-five percent of foreskin and 17% of fetal cord cells also showed trisomy 20, while no trisomic cells were detected in newborn blood. High frequency mosaicism for trisomy 20 in this case was thus due to true embryonic origin. Extensive counseling and prenatal follow-up in this case led to an unaffected liveborn, and guarded optimism may be warranted for future cases of mosaic trisomy 20 detected prenatally.

Cytogenetic abnormalities in an ossifying fibroma from a patient with bilateral retinoblastoma.

Cytogenetic analysis of a cemento-ossifying fibroma from a patient with nonfamilial bilateral multicentric retinoblastoma revealed three reciprocal translocations with the karyotype 46,XY,t(1;18)(q21;q21.3),t(3;10)(p13;q22),t(6;11)(p22;p15). Routine and high-resolution cytogenetic analysis of peripheral blood leukocytes showed an apparently normal, 46,XY chromosome pattern with no deletion of chromosome 13. Molecular analysis demonstrated no gross differences in the retinoblastoma gene or the TP53 gene between constitutional and tumor DNA. This is the first cytogenetic analysis of a cemento-ossifying fibroma and the first report of this tumor in a retinoblastoma patient. The data may be added to the small, but growing literature on cytogenetic aberrations in benign tumors and may lend insight into genes involved in cell proliferation and neoplastic transformation.

Chromosomal breakpoints in cholangiocarcinoma cell lines.

Little is known about the genetics and biology of cholangiocarcinoma (intrahepatic bile duct carcinoma). Only three human bile duct carcinoma cell lines have been described in the literature. We present the first detailed cytogenetic analysis of two cell lines; a new cell line designated PCI:SG231, established in our laboratory, and RPMI-7451, a previously established cell line. Both lines had highly aneuploid karyotypes with complex rearrangements including marker chromosomes. PCI:SG231, harvested after 50 days in culture, had a modal and median chromosome number of 65, and many cells contained double-minute chromosomes. RPMI-7451 had a modal and median chromosome number of 67. C-banding confirmed the presence of dicentric chromosomes in PCI:SG231. Q-banding confirmed the absence of the Y chromosome in PCI:SG231 and the presence of a der(1)t(Y;1)(q11;p11) chromosome in RPMI-7451. Numerical abnormalities common to both lines included trisomies 2, 5, 11, and 20. Chromosomes 1, 5, 7, and 12 were most commonly involved in structural abnormalities in both lines. Consistent chromosomal breakpoints included 7q22 and 12p11-12. PCI:SG231 was tumorigenic in immunosuppressed nude mice and was histologically similar to the original tumor. Additional cholangiocarcinoma cell lines are being developed to continue the study of the genetics and cell biology of this disorder.

The role of polyfusomes in generating branched chains of cystocytes during Drosophila oogenesis.

Three-dimensional models were constructed utilizing the information gained from electron micrographs of serial sections of two clones of cystocytes undergoing their terminal divisions. In each clone a polyfusome connected all eight cystocytes together. Each of the spindles was oriented so that one pole touched the polyfusomes, while the other pointed away from it. This positioning of spindles ensures that one cell of each dividing pair retains all previously formed canals, while the other receives none. The two cells that eventually come to contain the maximum number of canals and fusomal material are the ones that differentiate as pro-oocytes, while the others become nurse cells. The orientation of each spindle suggests that the polyfusome formed at one division determines the placement of the cytoskeletal fibers that anchor the spindles formed at the next division. There is a centripetal gathering together of new canals following each cycle of cystocyte division, which is thought to result from the subsequent contraction of the polyfusomal system. Females homozygous for the otu1 mutation are characterized by ovarian tumors, which result when germarial cystocytes undergo supernumerary divisions and fail to differentiate into either nurse cells or oocytes. An analysis of electron micrographs taken of serially sectioned, mutant germaria showed that most germ cells were single or belonged to clusters of two or three interconnected cells. Therefore otu1 cystocytes are unable to undergo a sustained series of arrested cleavages. These cystocytes contain fusomal material that shows ultrastructural differences from normal polyfusomes. We conclude: 1) that a normal polyfusomal system is a necessary prerequisite for the production of a branched chain of cystocytes and for their subsequent differentiation into pro-oocytes and nurse cells; and 2) that a product encoded by the otu+ gene is essential for the construction of a functional polyfusome.

Multiplicity of functions for the otu gene products during Drosophila oogenesis.

The ovarian tumor gene behaves as if it encodes a product (OGP), which is required during several early steps in the transformation of oogonia into functional oocytes. Seventeen ethyl methane sulfonate-induced mutations have been studied, and their mutant phenotypes can be explained as graded responses by individual germ cells to different levels of OGP synthesized by the mutant germ cells themselves. The lowest and highest levels of OGP appear to be produced by otu10 and otu14, respectively. The 15 mutants with intermediate OGP levels are temperature sensitive; subnormal temperatures improve ovarian development, while above-normal temperatures suppress it. A subgroup of these mutants are unable to form a system of actin microfilament bundles in the cortical cytoplasm of their nurse cells during stage 10B, and these defective nurse cells are unable to transport their cytoplasm to the oocyte, as normally happens between stages 10B and 12. In addition to its role in the actin-mediated transport of nurse cell cytoplasm, OGP also appears to alter the morphology of giant polytene chromosomes, which form as the nurse cells undergo endocycles of DNA replication. Genetic evidence suggests that otu also encodes a second product (SP) that is utilized late in oogenesis. SP is required for the synthesis in the ooplasm of glycogen-rich, beta yolk spheres. Products of the otu gene also play a vital but unknown role in embryogenesis.

Complementation between alleles at the ovarian tumor locus of Drosophila melanogaster.

The (ovarian tumor) otu gene resides at 23.2 on the genetic map of the X chromosome and near 7F1 on the cytological map. This germ line-expressed locus behaves as if it encodes a gene product which is required during certain steps in the transformation of oogonia into functional oocytes. On the basis of their ovarian morphologies 17 ethyl methane sulfonate (EMS)-induced mutants have been distributed among three developmental classes as follows: quiescent (eight), oncogenic (four), and differentiated (five). The otu13 and otu14 alleles interact to yield fertile females, and many other heteroallelic combinations show partial complementation. Since many mutant alleles interact beneficially, the functional product of the otu gene may be a multimer. We conclude, from an analysis of heteroallelic interactions and dosage effects, that the abnormal phenotypes observed are graded consequences of reduced levels of functional gene product and that the minimum concentration required for development increases as oogenesis proceeds.