Chromosomal abnormalities and p53 gene mutation in a cardiac angiosarcoma.

Angiosarcoma is the most common malignant neoplasm of the heart. However, to the authors' knowledge, no cytogenetic study of cardiac angiosarcoma has been reported. In the current study, an angiosarcoma from the right atrium of a 29-year-old man was investigated. Examination of tissue sections indicated that the tumor was a high grade epithelioid angiosarcoma of the heart. Cytogenetic analysis of tumor cells revealed a hyperdiploid clonal population with chromosomal numerical changes and one structural rearrangement, which was defined as: 55, XY, +der(1;17) (q10:q10), +2, +7, +8, +8, +19, +20, +21, +22. Multicolor fluorescent in situ hybridization on paraffin-embedded tissue sections illustrated polysomy of chromosome 8 in tumor cells. In addition, immunohistochemical analysis showed high expression of mutated p53 gene products in tumor cell nuclei. These findings demonstrate the involvement of chromosomal anomalies and gene mutation in cardiac angiosarcoma and suggest they play a role in neoplasia of the heart.

Transcriptional basis for the differences in inducible nitric oxide synthase (iNOS) expression between nonmetastatic and metastatic murine melanoma cell lines.

An inverse correlation exists between expression of the inducible nitric oxide synthase (iNOS) gene and the ability of cloned K1735 murine melanoma cell lines to metastasize. We have analyzed the basis for the difference in iNOS induction by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) in metastatic and non-metastatic K1735 cells. Nuclear run-on (NRO) assays revealed an upregulation of iNOS transcription on treatment with IFN-gamma plus LPS in nonmetastatic cells but not in a metastatic line. Transcription factors IFN regulatory factor 1 (IRF-1) and NF-kappaB were induced and functional in both metastatic and nonmetastatic K1735 lines treated with IFN-gamma plus LPS. Furthermore, a reporter construct driven by the wild-type iNOS promoter was transcriptionally activated in both nonmetastatic and metastatic cells. The iNOS-inducible phenotype was dominant in somatic cell hybrids generated by the fusion of nonmetastatic and metastatic cells, suggesting that no inhibitors of iNOS expression are present in metastatic cells. We conclude that the selective block in iNOS transcription in metastatic K1735 cells is likely due to an alteration in iNOS gene regulatory sequences. However, no such alteration was detected within the 1.7 kb iNOS promoter region in metastatic cells.

Chromosomal analysis of early fetal losses in relation to transvaginal ultrasonographic detection of fetal heart motion after infertility.

OBJECTIVE: To evaluate the prognostic value of transvaginal ultrasound (TVUS) detection of fetal heart motion (FHM) in view of maternal age and chromosomal analysis of spontaneously aborted fetal tissue. DESIGN: A 3-year retrospective, descriptive study. SETTING: Two medical center-based infertility-care facilities. PATIENT(S): 336 pregnancies were initiated by intrauterine insemination or embryo transfer for women of reproductive age who were seeking infertility treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): beta hCG levels measured > 40 mIU/mL at 4-5 weeks' gestation and were followed by an initial TVUS at 5-8 weeks. Of these pregnancies, 52 ended in a first trimester loss. Twenty were defined by failure to detect FHM by 7 weeks' gestation (Group I), and 32 were marked by the loss of prior FHM at a mean of 2.6 weeks later (Group II). Fetal tissue was removed by dilatation and suction curettage. Cytogenetic studies were performed from short-term cultures of dissected chorionic villi and/or sac. RESULT(S): Chromosomal aberrations were found in 75.0% of abortuses in Group I and 65.6% in Group II. Different types of chromosomal abnormalities were present in each these groups. The maternal age-related trisomies which can progress to term (i.e., 13, 18, 21) were associated with early TVUS detection of FHM. The frequency of chromosomal abnormalities varied significantly with maternal age, with normal fetal karyotypes in 7 of 11 (63.6%) women < 35 years, but only in 9 of 41 (22.0%) women > or = 35 years despite the detection of FHM in 24 of 41 (58.5%) of these older women. Detection of FHM was associated with pregnancies continuing beyond the first trimester in 284 of 316 (90.0%) overall, but differed significantly with age (166 of 174 [95.4%] women < 35 years vs. 118 of 142 [83.1%] women > or = 35 years). CONCLUSION(S): Although the occurrence of chromosomal abnormalities in spontaneous demises did not differ according to TVUS detection of FHM, the types of aberrations were distributed differently. Since maternal age remains a significant factor in early fetal loss, TVUS detection of FHM should not be as reassuring for women > or = 35 years as for younger women.

Numerical abnormalities of chromosomes 7, 18, and x in precancerous breast disease defined by fluorescent in situ hybridization.

Nonrandom numerical chromosomal abnormalities (NCA) are frequent in invasive breast cancer, but little is known about such changes in microscopic precursor lesions. Mammographically detected "suspicious" breast lesions were localized by specimen radiology of sliced breast tissue. The slices containing the lesion were imprinted onto coated slides by gentle scraping. The corresponding hematoxylin and eosin stained histologic sections and Diff-Quik stained imprints were used for classification as ductal hyperplasia (DH), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS). Additional slide imprints were evaluated for copy number of chromosomes 7, 18, and X by using fluorescent in situ hybridization with alpha satellite probes. NCA were detected in 1 of 9 (11%) cases of DH, in 2 of 8 (25%) cases of ADH, and in 14 of 16 (87%) cases of DCIS. There was selective loss (chromosome 18) in one case of DCIS; all other cases with NCA had a gain of at least one chromosome. There is a progressive increase in incidence of NCA in DH, ADH and DCIS. The majority of NCA are chromosomal gains.

Normal human mesothelial cells and mesothelioma cell lines express insulin-like growth factor I and associated molecules.

Insulin-like growth factor (IGF) I has important growth regulatory functions in normal growth and development. IGF-I is also a mitogen for a number of cancer cell lines; however, its autocrine effect has not been well established. In this study, the expression of IGF-I, its receptor, and its major serum-binding protein were examined in 5 normal human mesothelial (NHM) cell samples and 11 pleural mesothelioma cell lines. All NHM cells and mesothelioma cell lines expressed IGF-I, IGF-binding protein 3 (IGFBP-3), and IGF-I receptor mRNA by either Northern blot or reverse transcription polymerase chain reaction analysis. IGF-I (0.136 +/- 0.024 ng/ml, mean +/- SEM) and IGFBP-3 (18.5 +/- 3.2 ng/ml) proteins were readily detected in the conditioned medium of mesothelioma cell lines but were not greater than corresponding measurements in that of NHM cells (IGF-I, 0.120 +/- 0.080 ng/ml; IGFBP-3, 15.9 +/- 1.3 ng/ml). Exogenous recombinant IGF-I stimulated cell proliferation of NHM cells, demonstrating the presence of a functional IGF-I receptor. Our results suggest that IGF-I may function as an autocrine growth stimulus in normal proliferating mesothelial cells, which may contribute to their malignant transformation.

X-chromosome monosomy in the myelin-deficient rat mutant.

We have identified three examples of female Wistar rats exhibiting the tremor and seizures characteristic of the X-linked myelin deficiency (md) mutation, which is ordinarily seen only in males. Cytogenetic study of two of these animals has shown them to have 41 chromosomes instead of the normal 42. The missing chromosome was identified as an X chromosome by G-banding analysis. These animals thus have an XO genotype comparable to that in Turner's syndrome. Anatomically, one of the animals, which was studied in detail, showed no abnormality of the uterus, and the ovaries, although somewhat smaller than normal, were histologically indistinguishable from those in a normal female rat. No evidence of endocardial fibroelastosis was detected, nor was there any anomaly of the aorta. The myelin deficiency in the central nervous system was comparable to that in hemizygous mutant male rats. XO monosomy in the Wistar rat thus has little effect on phenotype and is more comparable to that in mice than to Turner's syndrome in man. The myelin-deficient rat is useful for studies of X-chromosome monosomy since XO females can readily be identified by the neurological syndrome characteristic of the md mutation.

Flow cytometry of fetal adrenal glands with adrenocortical cytomegaly.

Adrenal glands from four autopsied fetuses of 18 to 36 weeks gestation showed varying degrees of cortical cytomegaly. Formalin-fixed, paraffin-embedded sections from these four pairs of glands were studied by flow cytometry to analyze their DNA content and cell cycle parameters. Flow cytometry of Case 1, which had diffuse bilateral cytomegaly, demonstrated a major diploid peak, an increased percentage of tetraploid cells, and a decrease in S phase compared to an age-matched control with no evidence of cytomegaly (Case 2). Cases 3, 4, and 5 showed focal and/or unilateral adrenocortical cytomegaly and were diploid by flow cytometry with no differences in synthetic or tetraploid fractions compared to the control tissues. The focal distribution of the lesions or the limits of resolution of the instrumentation could account for some of these results. However, the findings in Case 1 suggest that the cytomegalic cells are tetraploid in DNA content and may have decreased DNA synthetic activity. A current hypothesis that these cells have undergone a period of sustained hyperactivity followed by exhaustion in reaction to an unknown stimulus is supported by our observations.

The karyotype of Philadelphia chromosome-negative, bcr rearrangement-positive chronic myeloid leukemia.

Philadelphia (Ph) chromosome negative chronic myeloid leukemia (CML) can be distinguished from clinically similar disorders on the basis of the presence of rearrangement of the breakpoint cluster region (bcr) of chromosome 22. We have identified six patients with Ph-negative CML, each with bcr rearrangement. Apparently normal karyotypes were observed in two cases, and a third contained a rearrangement that did not appear to involve chromosomes 9 or 22. The other three cases had translocations involving chromosome band 9q34 but no case contained the common derivative chromosome 9pter----9q34::22q11----22qter. One case appeared to contain either a deletion of an unrearranged bcr locus in approximately 50% of cells or duplication of rearranged bcr, both 5' and 3' of the chromosome 22 breakpoint. Considerable complexity exists in the types of genetic changes that can juxtapose bcr and the c-abl oncogene in CML. Based on the molecular and cytogenetic analyses of these and other cases described in the literature, we conclude that most cases of true Ph-negative CML arise from submicroscopic genetic exchanges rather than masking of simple t(9;22)(q34;q11) translocations by secondary rearrangements.

Cytogenetic diversity in primary human tumors.

Cytogenetic patterns from primary short-term culture of breast cancer, renal carcinoma, and tumors of the central nervous system are presented to illustrate the range of karyotypic diversity of human solid tumors as well as their biologic differences in culture systems that support their growth. These studies have illustrated several major issues. 1) Results vary with the tissue of origin: primary cultures from breast are almost uniformly diploid, while renal tumors are near-diploid, mosaic, and show clonal aberrations; and CNS tumors are heterogeneous: some diploid, some near-diploid and some highly aneuploid. 2) Results after short-term culture are selective, representing subpopulations from the heterogeneous cells that are detected on direct analysis of fresh tumors by cytogenetics or flow cytometry (FCM). It is not yet clear whether prognosis depends on the dominant population of the primary tumor or alternatively should be influenced by detection of small aneuploid subpopulations. 3) Evidence from all three tumor types supports the interpretation that cytogenetically normal diploid cells constitute part of some tumor populations, and may be better adapted to routine growth in culture than aneuploid subpopulations from the same primary tumors. These cells may also compose a major portion of the viable population of tumors in vivo and, therefore, could represent a useful model for studies of tumorigenesis and therapeutic regimens.

Prenatal diagnosis of a de novo complex chromosomal rearrangement involving four chromosomes.

A complex chromosome rearrangement, apparently a balanced translocation involving chromosomes 4, 6, 15 and 16, was found in cultured cells of amniotic fluid from a 32-year-old primigravida who requested amniocentesis for prenatal diagnosis because of a family history of mental retardation. Chromosome analysis of peripheral blood from both parents were normal. The couple was counselled for the prenatal diagnosis of this de novo complex translocation and, subsequently, elected to terminate the pregnancy. Post-mortem examination revealed a 23-week fetus with intrauterine growth retardation. The identical chromosome rearrangement was subsequently confirmed in cultured fibroblasts from skin and cord obtained from the abortus. To our knowledge, this is the first report where routine prenatal diagnosis revealed a fetus with a balanced complex chromosomal rearrangement involving four chromosomes of de novo origin.

Acute nonlymphocytic leukemia following gastric adenocarcinoma treated by surgery alone. Evidence for etiologic heterogeneity of "secondary" leukemias.

The case of a 36-year-old Hispanic man who developed acute nonlymphocytic leukemia 18 months following gastric adenocarcinoma treated by surgery alone is presented. Cytogenetic analysis of the leukemic cells revealed numerical and structural chromosomal rearrangements including chromosomes 5 and 7 and immunologic characterization of the blasts revealed terminal deoxynucleotidyltransferase positivity with monocytoid features. This report suggests that not all cases of acute nonlymphocytic leukemia following chemotherapy and/or radiotherapy, which characteristically display similar cytogenetic and immunologic features, should be exclusively ascribed to the leukemogenic properties of anticancer treatment.

Philadelphia-chromosome-negative chronic myelogenous leukemia with incomplete trisomy 1q following chemotherapy for ovarian carcinoma.

A 59-year-old woman was treated with surgery followed by monthly injections of the alkylating agent thiotepa for a granulosa cell tumor of the left ovary. Chemotherapy was continued for 22 years. At the age of 84, chronic myelogenous leukemia (CML) developed. Cytogenetic studies revealed incomplete trisomy of the long arm of chromosome No. 1 as the only karyotypic abnormality. No Philadelphia chromosome was detected. The significance of trisomy 1q as an isolated cytogenetic abnormality in CML and the occurrence of CML following treatment of ovarian cancer are discussed.

High resolution analysis of the timing of replication of specific DNA sequences during S phase of mammalian cells.

A new method, utilizing selective photodegradation of 5-bromo-deoxyuridine (BUdR)-substituted DNA and flow cytometry, has been developed for analyzing the timing of replication of specific DNA sequences. Chemically synchronized Chinese hamster ovary cells were given a pulse of the deoxythymidine analogue, BUdR, at different times during S phase, and flow sorted according to DNA content, before DNA isolation. Newly-replicated, unifilarly BUdR-substituted DNA was selectively degraded by treatment with 33258 Hoechst plus near UV light followed by S1 nuclease digestion; the resistant DNA was analyzed for its content of 18s and 28s rDNA or dihydrofolate reductase (DHFR) sequences via Southern blot analysis. Both the rDNA and DHFR sequences were found to replicate almost entirely during the first quarter of S phase. The approach described should have general utility for analyzing replication kinetics of specific DNA sequences in mammalian cells.

Developmentally regulated nonhistone proteins: evidence for deoxyribonucleic acid binding role and localization near deoxyribonuclease I sensitive domains of precartilage cell chromatin.

Differentiation of cartilage from precartilage mesenchyme in the chick embryo is accompanied by the loss of two abundant nonhistone proteins (Mr 35 500 and 125 000) termed PCP 35.5 and PCP 125. Here we examine the distribution of these and other developmentally regulated nonhistones in nuclease-sensitive regions of precartilage and cartilage chromatin. In particular, we show that PCP 35.5 is a tight DNA-binding protein that is localized near deoxyribonuclease I (DNase I) sensitive regions of precartilage chromatin. Localization of nonhistones was demonstrated by excising domains of precartilage chromatin with DNase II which are simultaneously highly enriched in PCP 35.5, in PCP 125, and DNase I sensitive DNA sequences. These domains comprise at least 25% of the cell's DNase I sensitive sequences, as well as small DNase I resistant regions with which the two nonhistones are associated. These findings suggest that PCP 35.5 (and possibly PCP 125) may play a developmentally regulated role nearby DNase I sensitive domains of the cartilage progenitor cell chromatin.

Talpid2 mutant of the chicken with perturbed cartilage development has an altered precartilage-specific chromatin protein.

An abundant nonhistone protein (Mr, 125,000) is lost from the chromatin embryonic chicken precartilage mesenchyme cells as they differentiate into cartilage [Newman, SA., Birnbaum, J & Yeoh, G.C.T.(1976) Nature (London) 259, 417-418]. We have now examined the chromatin proteins of precartilage and cartilage cells of chicken embryos carrying the talpid2 gene which causes a perturbed patern of cartilage differentiation in the homozygous state. We find that homozygous talpid2 precartilage chromatin differs from that of the normal cell type in having its abundant precartilage chromatin protein decreased to a Mr of approximately 120,000 and in having a "precocious" cartilage-like pattern of proteins in the Mr 35,500-36,500 region. The precartilage chromatin of talpid2 heterozygotes is completely normal within the resolution of our techniques, as is the cartilage chromatin of the homozygote and heterozygote talpid2 embryos. The correlation of an aberration in a developmentally significant chromatin protein with the perturbed development of its tissue of origin is discussed.