The last 20 years have witnessed an astounding evolution of cytogenetic approaches to cancer diagnosis and prognostication. Molecular techniques and, in particular, nonisotopically-labeled nucleic acid probes and fluorescence in situ hybridization (FISH)-based techniques have replaced the costly and potentially dangerous radioactive techniques used in research and the clinical detection of genetic alterations in tumor cells. Fluorescent DNA probes also enabled the screening for very subtle chromosomal changes. Clinical laboratories now choose from a growing number of FISH-based cytogenetic tests to support physician's diagnoses of the causes and the course of a disease. Depending on the specimen, state-of-the-art FISH techniques allow the localization and scoring of 10–24 different targets and overcome previous problems associated with target colocalization and detection system bandwidth. FISH-based analyses have been applied very successfully to the analysis of single cells and have demonstrated the existence of cell clones of different chromosomal make-up within human tumors. This information provides disease-specific information to the attending physician and should enable the design of patient-specific protocols for disease intervention.

Chromosome translocations occur at a frequency of about 0.1% in the general population. Translocation carriers experience a higher incidence of infertility and reproductive failures and also a higher risk of conceiving chromosomally abnormal offspring. Patients who are carriers of Robertsonian or reciprocal translocations may benefit from in vitro fertilization (IVF) followed by preconception and preimplantation genetic diagnosis (PGD). Three approaches for PGD of structural abnormalities based on fluorescence in situ hybridization (FISH) are discussed in this chapter: chromosome painting of polar bodies, probes mapping distal to the break points, and probes spanning the break points. When the carrier is female, the chromosomally normal versus abnormal oocytes can be determined by using whole chromosome painting probes hybridized to the first polar body chromosomes. When the carrier is male, the only method available for PGD is the blastomere biopsy after in vitro fertilization (IVF) and in vitro culture of the embryo. The chapter describes rapid and inexpensive procedures to prepare case-specific probes for FISH-based PGD in germ cells or embryos by using yeast artificial chromosomes (YACs) as probes spanning or flanking translocation break points.

Chromosome abnormalities are common causes of congenital malformations and spontaneous abortions. They include structural abnormalities, polyploidy, trisomy, and mosaicism. In in vitro fertilization (IVF) programs, preimplantation genetic diagnosis (PGD) of oocytes and embryos has become the technique of choice to select against abnormal embryos before embryo transfer. For diagnosis of structural abnormalities, we developed case-specific breakpoint-spanning DNA probes. Screening of an in-house yeast artificial chromosome (YAC) library is facilitated by information from publicly available databases and published articles. Most numerical chromosome abnormalities, on the other hand, are detrimental to early embryonic development and increase with maternal age. We therefore developed a multichromosome screening technique based on spectral imaging to simultaneously detect and score as many as 10 different chromosome types. The probe set was chosen to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions. Detecting structural and numerical abnormalities in single interphase cells using spectral imaging is a powerful technique for multilocus genetic screening.

Numerical chromosome aberrations are incompatible with normal human development. Our laboratories develop hybridization-based screening tools that generate a maximum of cytogenetic information for each polar body or blastomere analyzed. The methods are developed considering that the abnormality might require preparation of case-specific probes and that only one or two cells will be available for diagnosis, most of which might be in the interphase stage. Furthermore, assay efficiencies have to be high, since there is typically not enough time to repeat an experiment or reconfirm a result prior to fertilization or embryo transfer. Structural alterations are delineated with breakpoint-spanning probes. When screening for numerical abnormalities, we apply a Spectral Imaging-based approach to simultaneously score as many as ten different chromosome types in individual interphase cells. Finally, DNA micro-arrays are under development to score all of the human chromosomes in a single experiment and to increase the resolution with which micro-deletions can be delineated.

In vitro model cell systems are important tools for studying mechanisms of radiation‐induced neoplastic transformation of human epithelial cells. In our study, the human thyroid epithelial cell line HTori‐3 was analyzed cytogenetically following exposure to different doses of α‐ and γ‐irradiation and subsequent tumor formation in athymic nude mice. Combining results from G‐banding, comparative genomic hybridization, and spectral karyotyping, chromosome abnormalities could be depicted in the parental line HTori‐3 and in nine different HTori lines established from the developed tumors. A number of chromosomal aberrations were found to be characteristic for simian virus 40 immortalization and/or radiation‐induced transformation of human thyroid epithelial cells. Common chromosomal changes in cell lines originating from different irradiation experiments were loss of 8q23 and 13cen‐q21 as well as gain of 1q32‐qter and 2q11.2‐q14.1. By comparison of chromosomal aberrations in cell lines exhibiting a different tumorigenic behavior, cytogenetic markers important for the tumorigenic process were studied. It appeared that deletions on chromosomes 9q32‐q34 and 7q21‐q31 as well as an increased copy number of chromosome 20 were important for the tumorigenic phenotype. A comparative breakpoint analysis of the marker chromosomes found and those observed in radiation‐induced childhood thyroid tumors from Belarus revealed a coincidence for a number of chromosome bands. Thus, the data support the usefulness of the established cell system as an in vitro model to study important steps during radiation‐induced malignant transformation in human thyroid cells. © 2001 Wiley‐Liss, Inc.

The microarray format of RNA transcript analysis should provide new clues to carcinogenic processes. Because of the complex and heterogeneous nature of most tumor samples, histochemical techniques, particularly RNA fluorescent in situ hybridization (FISH), are required to test the predictions from microarray expression experiments. Here we describe our approach to verify new microarray data by examining RNA expression levels of five to seven different transcripts in a very few cells via FISH.

Numerical chromosome aberrations are detrimental to early embryonic, fetal and perinatal development of mammals. When fetuses carrying a chromosomal imbalance survive to term, an aberrant gene dosage typically leads to stillbirth or causes a severely altered phenotype. Aneuploidy of any of the 24 chromosomes will negatively impact on human development, and a preimplantation and prenatal genetic diagnosis test should thus score as many chromosomes as possible. Since cells available for analysis are likely to be in interphase, we set out to develop a rapid enumeration procedure based on hybridization of chromosome-specific probes and spectral imaging detection. The probe set was chosen to allow the simultaneous enumeration of ten chromosome types and was expected to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions, i.e., human chromosomes 9, 13, 14, 15, 16, 18, 21, 22, X, and Y. Cell fixation protocols were optimized to achieve the desired detection sensitivity and reproducibility. We were able to resolve and identify ten separate chromosomal signals in interphase nuclei from different types of cells, including lymphocytes, uncultured amniocytes, and blastomeres. In summary, this study demonstrates the strength of spectral imaging, allowing us to construct partial spectral imaging karyotypes for individual interphase cells by assessing the number of each of the target chromosome types.

Genetic factors affecting postnatal γ-globin expression—a major modifier of the severity of both β-thalassemia and sickle cell anemia—have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human γ-globin. To model the human β-cluster in mice, with the goal of screening for loci affecting human γ-globin expression in vivo, we introduced a human β-globin cluster YAC transgene into the genome of FVB/N mice. The β-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) γ allele, resulting in postnatal expression of human γ-globin in transgenic mice. The level of human γ-globin for various F1 hybrids derived from crosses between the FVB/N transgenics and other inbred mouse strains was assessed. The γ-globin level of the (C3HeB/FeJ × FVB/N)F1 transgenic mice was noted to be significantly elevated. To map genes affecting postnatal γ-globin expression, we performed a 20-centiMorgan (cM) genome scan of a (C3HeB/FeJ × FVB/N)F1 transgenics × FVB/N backcross, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within an 18-cM interval of mouse Chromosome (Chr) 1 (LOD = 4.3) that contributes 10.9% of variation in γ-globin level. Combining transgenic modeling of the human β-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human γ-globin level in vivo.

Objectives: Numerical chromosome aberrations are detrimental to embryo survival and thus cause spontaneous abortions. We used fluorescence in situ hybridization (FISH) to determine the incidence of aneuploidy for chromosomes 1, 16, 18, and 21 in human eggs that failed to fertilize following either IVF or ICSI.Design: A total of 201 failed fertilized eggs from 91 patients were available for this study. Chromosome-specific aneuploidy, including both hypoploidy (nullisomy/monosomy) and hyperploidy (trisomy/tetrasomy), was determined for each of the four chromosomes with respect to patient age (<35 yrs, 35–39 yrs, >39 yrs).Materials and Methods: Institutional Review Board Approval was obtained for this study. Following fixation with ethanol:glacial acetic acid (3:1, v/v) and Wright's staining, suitable spreads were destained in methanol, pretreated with 2×SSC and pepsin, then post-fixed in 1% formaldehyde. The DNA denaturation was performed in 70% formamide/2×SSC at 75°C. DNA probes were purchased (CEP18, Vysis) or prepared in-house (pUC1.77, RMC16L001, LSP21). Slides were hybridized for 40–48 hr with probe mixture. Following removal of unbound probes, signals were scored by fluorescence microscopy. Statistical analyzes were performed using Chi square and Fisher's Exact Tests.Results: A total of 163 spreads were considered suitable for FISH analysis. Informative results were obtained from 114 spreads. There was a statistically significant age-related difference in aneuploidy frequency for chromosome 16 (13.2, 30 and 40% in the three age groups, respectively; P<0.05). Overall aneuploidy rates for chromosomes 1, 18 and 21 were 19.3, 18.8 and 24.3%, respectively, and did not show significant age-dependent increases. Frequencies for hypo- vs. hyperploidy showed chromosome-specific differences (% hypoploidy/total aneuploidy): 1: 59.1%; 16: 57.7%; 18: 66.7%; 21: 69.2%, although these differences did not reach statistical significance.Conclusion: There was an overall high frequency of aneuploidy (∼20%) among failed fertilized human eggs (including chromosome 1) and an age-related increase for chromosome 16. The tendency towards a higher ratio of hypoploidy for chromosomes 18 and 21 could represent a loss either of chromatids during fixation or of signals during FISH for these smaller chromosomes, or a loss of chromatids during meiosis I in the oocyte itself. These findings contribute to our understanding of the origin and fate of aneuploidy in humans. Supported by UC BioSTAR Grant and UCSF/Stanford Enterprise Fund (RAP) and BWH Ob/Gyn IVF Discretionary Funds (CR). Objectives: Numerical chromosome aberrations are detrimental to embryo survival and thus cause spontaneous abortions. We used fluorescence in situ hybridization (FISH) to determine the incidence of aneuploidy for chromosomes 1, 16, 18, and 21 in human eggs that failed to fertilize following either IVF or ICSI. Design: A total of 201 failed fertilized eggs from 91 patients were available for this study. Chromosome-specific aneuploidy, including both hypoploidy (nullisomy/monosomy) and hyperploidy (trisomy/tetrasomy), was determined for each of the four chromosomes with respect to patient age (<35 yrs, 35–39 yrs, >39 yrs). Materials and Methods: Institutional Review Board Approval was obtained for this study. Following fixation with ethanol:glacial acetic acid (3:1, v/v) and Wright's staining, suitable spreads were destained in methanol, pretreated with 2×SSC and pepsin, then post-fixed in 1% formaldehyde. The DNA denaturation was performed in 70% formamide/2×SSC at 75°C. DNA probes were purchased (CEP18, Vysis) or prepared in-house (pUC1.77, RMC16L001, LSP21). Slides were hybridized for 40–48 hr with probe mixture. Following removal of unbound probes, signals were scored by fluorescence microscopy. Statistical analyzes were performed using Chi square and Fisher's Exact Tests. Results: A total of 163 spreads were considered suitable for FISH analysis. Informative results were obtained from 114 spreads. There was a statistically significant age-related difference in aneuploidy frequency for chromosome 16 (13.2, 30 and 40% in the three age groups, respectively; P<0.05). Overall aneuploidy rates for chromosomes 1, 18 and 21 were 19.3, 18.8 and 24.3%, respectively, and did not show significant age-dependent increases. Frequencies for hypo- vs. hyperploidy showed chromosome-specific differences (% hypoploidy/total aneuploidy): 1: 59.1%; 16: 57.7%; 18: 66.7%; 21: 69.2%, although these differences did not reach statistical significance. Conclusion: There was an overall high frequency of aneuploidy (∼20%) among failed fertilized human eggs (including chromosome 1) and an age-related increase for chromosome 16. The tendency towards a higher ratio of hypoploidy for chromosomes 18 and 21 could represent a loss either of chromatids during fixation or of signals during FISH for these smaller chromosomes, or a loss of chromatids during meiosis I in the oocyte itself. These findings contribute to our understanding of the origin and fate of aneuploidy in humans. Supported by UC BioSTAR Grant and UCSF/Stanford Enterprise Fund (RAP) and BWH Ob/Gyn IVF Discretionary Funds (CR).

Balanced reciprocal translocations are known to interfere with homolog pairing in meiosis. Many individuals carrying such chromosomal abnormalities suffer from reduced fertility or spontaneous abortions and seek help in the form of assisted reproductive technology. Although most translocations are relatively easy to detect in metaphase cells, the majority of embryonic cells biopsied in the course of in vitro fertilization (IVF) procedures are in interphase. These nuclei are, thus, unsuitable for analysis by chromosome banding or painting using fluorescence in situ hybridization (FISH). Our assay, based on FISH detection of breakpoint-spanning DNA probes, identifies translocations in interphase nuclei by microscopic inspection of hybridization domains. Probes are selected that span the breakpoint regions on normal homologs. The probes should hybridize to several hundred kilobases of DNA flanking the breakpoint. The two breakpoint-spanning DNA probes for the translocation chromosomes are labeled in separate colors (e.g., red and green). The translocation event producing two fused red/green hybridization domains can then be detected in interphase cell nuclei using a fluorescence microscope. We applied this scheme to analyze somatic and germ cells from 21 translocation patients, each with distinct breakpoints. Here, we summarize our experience and provide a description of strategies, cost estimates, as well as typical time frames.

Spectral Imaging (SIm) has dramatically improved our ability to localize and quantitatively analyze multiple nucleic acid targets such as chromosomes, genes and gene transcripts. Studies on metaphase cells such as 'Spectral Karyotyping (SKY)' are less complicated than interphase cell studies because the objects (chromosomes) are spatially separated and ratio-labeled probes can be used to uniquely stain each chromosome type. Our research, however, targets the extensive cytogenetic and phenotypic analysis of interphase cells. The complex organization of interphase chromatin and co-localization of gene transcripts (RNAs) in nuclear or cytoplasmic domains requires unique fluorochrome-labeling for each nucleic acid target. An increasing number of commercially available dyes for probe labeling and software to deconvolute partially overlapping emission spectra has helped to overcome most of these obstacles. This presentation summarizes our experience in analyzing numerical and structural alterations in various human cell types (leukocytes, amniocytes, blastomeres or solid tissue) as well as our approach to multi-gene expression profiling using Sim. Examples illustrate a wide spectrum of groundbreaking techniques for interphase cell analysis. We demonstrate how ten or more chromosomes can be scored in interphase nuclei or the relative level of expression of different transforming RNAs in tumor cells can be measured by SIm.

Objective: To review 35 cases of preimplantation genetic diagnosis (PGD) of translocations with several methods, including telomeric probes.Design: Retrospective study.Setting: Clinical IVF laboratory.Patient(s): Thirty-five couples with one partner carrying a chromosomal translocation.Intervention(s): PGD of translocation after polar-body or embryo biopsy.Main Outcome Measure(s): Pregnancy outcome.Result(s): Several trends were observed. First, PGD can achieve a statistically significant reduction in spontaneous abortion, from 95% to 13%. Second, the chances of achieving pregnancy are correlated with 50% or more of the embryos being chromosomally normal. Third, patients with robertsonian translocations produced fewer abnormal gametes and more pregnancies than did patients with reciprocal translocations. Fourth, a new fluorescence in situ hybridization protocol for PGD of translocations, which involves applying telomeric probes, has proved adequately reliable with a 6% average error rate.Conclusion(s): PGD of translocations achieves a statistically significant reduction in spontaneous abortion, both for polar-body and blastomere biopsy cases. Pregnancy outcome depended on the number of normal embryos available for transfer, with patients having <50% abnormal embryos achieving the most pregnancies. Because robertsonian translocations caused fewer abnormal embryos than reciprocal translocations, they also resulted in higher rates of implantation. ((C) 2000 by American Society for Reproductive Medicine.)

Numerical aberrations involving parts of or entire chromosomes have detrimental effects on mammalian embryonic, and perinatal development. Only few fetuses with chromosomal imbalances survive to term, and their abnormalities lead to stillbirth or cause severely altered phenotypes in the offspring (such as trisomies involving chromosomes 13, 18, 21, and anomalies of X, and Y). Because aneuploidy of any of the 24 chromosomes will have significant consequences, an optimized preimplantation and prenatal genetic diagnosis (PGD) test will score all the chromosomes. Since most cells to be analyzed will be in interphase rather than metaphase, we developed a rapid procedure for the analysis of interphase cells such as lymphocytes, amniocytes, or early embryonic cells (blastomeres). Our approach was based on in situ hybridization of chromosome-specific non-isotopically labeled DNA probes and Spectral Imaging.The Spectral Imaging system uses an interferometer instead of standard emission filters in a fluorescence microscope to record high resolution spectra from fluorescently stained specimens. This bio-imaging system combines the techniques of fluorescence optical microscopy, charged coupled device imaging, Fourier spectroscopy, light microscopy, and powerful analysis software. The probe sets used here allowed simultaneous detection of 10 chromosomes (9, 13, 14, 15, 16, 18, 21, 22, X, Y) in interphase nuclei. Probes were obtained commercially or prepared in-house. Following 16-40h hybridization to interphase cells and removal of unbound probes, image spectra (range 450-850 nm, resolution 10 nm) were recorded and analyzed using an SD200 Spectral Imaging system (ASI, Carlsbad, CA). Initially some amniocytes were unscoreable due to their thickness, and fixation protocols had to be modified to achieve satisfactory results. In summary, this study shows the simultaneous detection of at least 10 different chromosomes in interphase cells using a novel approach for multi-chromosome analysis.

Thyroid carcinoma incidence is increased significantly after ionizing irradiation; however, the possible mechanisms have not yet been identified. To provide clues for an understanding of the radiation-induced transformation of thyroid epithelium, we analyzed the karyotypes of 56 childhood thyroid tumors that appeared in Belarus after the Chernobyl nuclear accident in 1986. We also studied eight secondary thyroid tumors that developed after radiotherapy. Metaphase preparations obtained from primary cultures were analyzed by G-banding. Clonal structural aberrations were found in 13 of 56 Belarussian cases and in 6 of 8 secondary tumors that developed after radiotherapy. Furthermore, we detected multiple chromosomal aberrations as well as complex rearrangements in some of these tumors and performed a detailed analysis of marker chromosomes from a single case using spectral karyotyping and comparative genomic hybridization in a childhood tumor from Belarus with a near-triploid karyotype. Both comparative genomic hybridization and spectral karyotyping analysis revealed structural alterations affecting identical chromosomes 1, 2, 9, and 13, among others. In addition to the known hot spots of alterations in papillary thyroid carcinomas on chromosomes 1q and 10q, a comprehensive breakpoint analysis in the pooled data set revealed novel breakpoints on chromosomes 4q, 5q, 6p, 12q, 13q, and 14q. The chromosomal aberrations in these tumors may provide suitable starting points for the positional cloning of genes involved in radiation-induced tumorigenesis.

Radiocarcinogenesis is widely recognized as occupational, environmental and therapeutical hazard, but the underlying mechanisms and cellular targets have not yet been identified. We applied SKY to study chromosomal rearrangements leading to malignant transformation of irradiated thyroid epithelial cells.SKY is a recently developed technique to detect translocations involving non-homologous based on unique staining of all 24 human chromosomes by hybridization with a mixture of whole chromosome painting probes. A tuneable interferometer mounted on a fluorescence microscope in front of a CCD camera allows to record the 400nm-1000nm fluorescence spectrum for each pixel in the image. After background correction, spectra recorded for each pixel are compared to reference spectra stored previously for each chromosome-specific probe. Thus, pixel spectra can be associated with specific chromosomes and displayed in 'classification' colors, which are defined so that even small translocations become readily discernible.SKY analysis was performed on several radiation-transformed cell lines. Line S48T was generated from a primary tumor of a child exposed to elevated levels of radiation following the Chernobyl nuclear accident. Subclones were generated from the human thyroid epithelial cell line (HTori-3) by exposure to gamma or alpha irradiation. SKY analysis revealed multiple translocations and, combined with G-banding, allowed the definition of targets for positional cloning of tumor related genes.