Interphase cytogenetics in pathology: principles, methods, and applications of fluorescence in situ hybridization (FISH)

 Characteristic chromosome aberrations have been identified in various tumors. Fluorescence in situ hybridization (FISH) using specific probes that are generated by vector cloning or in vitro amplification and labeled with fluorescent dyes allow for the detection of these genetic changes in interphase cells. This technique, that is also referred to as ”interphase cytogenetics”, can be performed in cytological preparations as well as in sections of routinely formaldehyde-fixed and paraffin-embedded tissue. In cancer research and diagnostics, interphase cytogenetics by FISH is used to detect numerical chromosome changes and structural aberrations, e.g., translocations, deletions, or amplifications. In this technical overview, we explain the principles of the FISH method and provide protocols for FISH in cytological preparations and paraffin sections. Moreover, possible applications of FISH are discussed. Accepted: 22 July 1997     

Fluorescence in situ hybridization: applications in cytogenetics and gene mapping

Unique sequences, chromosomal subregions, or entire genomes can be specifically highlighted in metaphase or interphase cells by fluorescence in situ hybridization (FISH). This technique can be used to identify chromosomes, detect chromosomal abnormalities or determine the chromosomal location of specific sequences. FISH plays an increasingly important role in a variety of research areas, including cytogenetics, prenatal diagnosis, tumor biology, gene amplification and gene mapping.     

Molecular cytogenetics in metaphase and interphase cells for cancer and genetic research, diagnosis and prognosis. Application in tissue sections and cell suspensions

As the pioneer among molecular cytogenetics techniques, fluorescence in situ hybridization (FISH) allows identification of specific sequences in a structurally preserved cell, in metaphase or interphase. This technique, based on the complementary double-stranded nature of DNA, hybridizes labeled specific DNA (probe). The probe, bound to the target, will be developed into a fluorescent signal. The fact that the signal can be detected clearly, even when fixed in interphase, improves the accuracy of the results, since in some cases it is extremely difficult to obtain mitotic samples. FISH is still used mostly in research, but there are diagnostic applications. New nomenclature is being developed in order to define many of the aberrations that were not distinguished before FISH. Prenatal diagnosis of aneuploidies and malignancies are promptly detected with FISH, which is very useful in critical cases. In some tumors, where chromosomal abnormalities are too complicated to classify manually, the technique of comparative genomic hybridization (CGH), a competitive FISH, allows examiners to determine complete or partial gain or loss of chromosomes. CGH results allow the classification of many tumor cell lines and along with other complementary techniques, like microdissection-FISH, PRINS, etc., increase the possibility of choosing an appropriate treatment for cancer patients.     

Combined RxFISH/G-banding allows refined karyotyping of solid tumors

Chromosome banding analysis of solid tumors often yields incomplete karyotypes because of the complex rearrangements encountered. The addition of fluorescence in situ hybridization (FISH) methods has helped improve the accuracy of solid tumor cytogenetics, but the absence of screening qualities from standard FISH approaches has proved a severe limitation. We describe the cytogenetic analysis of ten solid tumors using G-banding followed by cross-species color banding (RxFISH), a FISH-based screening technique giving a chromosome-specific banding pattern based on the genomic homologies between humans and gibbons. The addition of RxFISH analysis in all cases led to the identification of previously unidentified intra- as well as interchromosomal rearrangements, thus giving a much more certain and detailed karyotype. In two gastric stromal sarcomas, a tumor type for which no cytogenetic data were hitherto available, numerical chromosomal aberrations dominated, but one of the tumors also carried an unbalanced 7;17-translocation with the same breakpoint in chromosome 17 as that seen in endometrial stromal sarcomas. Received: 15 January 1999 / Accepted: 5 March 1999     

Cytogenetic analysis of human solid tumors by in situ hybridization with a set of 12 chromosome-specific DNA probes

We have applied fluorescent in situ hybridization (FISH) to assess the presence of numerical chromosome aberrations in fresh specimens of human solid tumors of varying histology. For this purpose, a set of 12 biotinylated chromosome-specific, repetitive alpha-satellite DNA probes (for chromosomes 1, 6, 7, 9, 10, 11, 15, 16, 17, 18, X and Y) were hybridized directly to isolated interphase nuclei. Utilizing this approach, we found numerical chromosome changes in all tumors. FISH ploidy profiles were in accordance with flow cytometric DNA histograms of these tumor cells.     

Fluorescence in situ hybridization (FISH) on human chromosomes using photoprobe biotin-labeled probes.

Fluorescence in situ hybridization (FISH) on human chromosomes in meta- and interphase is a well-established technique in clinical and tumor cytogenetics and for studies of evolution and interphase architecture. Many different protocols for labeling the DNA probes used for FISH have been published. Here we describe for the first time the successful use of Photoprobe biotin-labeled DNA probes in FISH experiments. Yeast artificial chromosome (YAC) and whole chromosome painting (wcp) probes were tested.     

Comparison of metaphase and interphase FISH monitoring of minimal residual disease with MLL gene probe: case study of AML with t(9;11).

The place of FISH in the monitoring of minimal residual disease (MRD) is yet to be fully characterised. Routine bone marrow cytogenetics at diagnosis in a 22 year old patient with acute myeloid leukemia FAB type M5 detected a translocation t(9;11)(p22;q23). We report our investigations to assess residual levels of translocation using a FISH probe designed to detect a gene split by the translocation. We used MLL (Oncor), a probe which spans the MLL gene at 11q23, in both metaphase and interphase preparations. At diagnosis, metaphase FISH showed 3 distinct cell lines-normal with 2 signals, abnormal with 3 signals and abnormal with 2 signals, while interphase FISH showed only 2 cell lines, one with 2 signals (which could be normal or abnormal) and one with 3 signals (split MLL). Following treatment, with the patient in clinical remission, 7 further cytogenetic analyses and 2 further FISH analyses were compared. Our results suggest that monitoring of the t(9;11) by metaphase FISH is feasible and straightforward compared to cytogenetics but interphase FISH may be problematic.     

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.     

Detection of chromosome aberrations in paraffin sections of seven gonadal yolk sac tumors of childhood

Yolk sac tumors are the most frequent kind of malignant pediatric germ cell tumor and may have a fundamentally different pathogenesis than adult germ cell tumors. Since few cytogenetic studies have been performed so far, in situ hybridization was applied to interphase cell nuclei of seven gonadal yolk sac tumors of childhood in routine paraffin-embedded tissue sections. The panel of chromosome-specific DNA probes was selected on the basis of their relevance in adult germ cell tumors and consisted of five DNA probes specific for the (peri)centromeric regions of chromosomes 1, 8, 12, 17 and/or X and/ or one DNA probe specific for the subtelomeric region of chromosome 1 (p36.3). As in adult germ cell tumors, all pediatric gonadal yolk sac tumors had an increased incidence of numerical chromosome aberrations. All tumors showed an overrepresentation of at least three chromosomes. Gains of chromosome 12, which is highly specific in adult germ cell tumors, were diagnosed in six pediatric gonadal yolk sac tumors. The DNA indices determined in the paraffin-embedded tumor material correlated well with the in situ hybridization findings. A chromosome was either over- or underrepresented, compared with the corresponding DNA indices, in only a few cases. The short arm of chromosome 1 in adult germ cell tumors is often involved in structural aberrations. In pediatric germ cell tumors, the short arm of chromosome 1 is also a nonrandom site of structural aberrations. Moreover, the presence of a deletion at 1p36.3 in four out of five tumors suggests that the loss of gene(s) in this region is an important event in the pathogenesis of gonadal yolk sac tumors of childhood.     

FISH panels for hematologic malignancies

Cytogenetic analysis of hematological malignancies has played a crucial role in the diagnosis and clinical management of patients, as well as in providing fundamental insights into the genetic basis of the pathogenesis of these diseases. Leukemias and lymphomas have lent themselves readily to karyotypic analysis and undoubtedly represent the greatest successes of cytogenetics in human cancer. Several cytogenetic changes have been shown to have considerable prognostic significance also and are being used as measurable targets for response to therapy. Molecular characterization of the recurrent cytogenetic abnormalities has identified genes involved in leukemogenesis and formed a basis for specific treatment strategies. Fluorescence in situ hybridization (FISH) analysis, since its introduction, has revolutionized the field and enabled a more precise determination of the presence and frequency of genetic abnormalities. It is particularly indispensable where metaphase cytogenetics may be difficult in the largely quiescent cells of some hematological malignancies, particularly the lymphoid disorders. FISH probes have been used extensively to detect nonrandom abnormalities in interphase nuclei and the true incidence of chromosome abnormalities has been proven to be much higher than that detected by conventional chromosomal analysis. The avail- ability of a comprehensive line of commercial probes for rapid identification of critical genetic aberrations has contributed to the widespread use of this technique. It has also led to the current practice in most laboratories to test for genetic aberrations by using FISH panels that have been designed to detect genetic changes important not only in the diagnosis of leukemias and lymphomas, but also because of their association with prognosis, to identify high-risk populations in specific hematological cancers, so they can be targeted for aggressive therapy.     

Variations in alphoid DNA sequences escape detection of aneuploidy at interphase by FISH technique.

The advent of a new staining technique, termed fluorescence in situ hybridization (FISH), allows the rapid identification of the genomic constitution of an individual with aneuploidy even in interphase nuclei through the use of a series of chromosome-specific DNA probes, an approach termed "interphase cytogenetics." However, alphoid DNA sequences of every centromere are polymorphic (heteromorphic), and the number of targeted sequences may be below the detection level of a specific DNA probe, thus escaping detection and resulting in the imprecise identification of the chromosomal constitution at interphase. The limitations associated with the FISH technique have dire consequences which are emphasized here with an example in which the presence of an additional chromosome 21 in two siblings born consecutively with trisomy 21 (Down syndrome) was not detected by "interphase cytogenetics." The copy number of alphoid DNA sequences of one of the paternal chromosomes 21 was low and resulted in discordance between domain numbers at interphase and actual chromosome numbers at metaphase in both children. This is an isolated incident that could have led to a misdiagnosis if FISH were the only test employed. Although the advantages of this technology are undeniably enormous, the present finding has made it apparent that precise standards and reliability of the procedure must be established prior to its routine application.     

荧光原位杂交技术及其在植物研究中的应用

荧光原位杂交(FISH)是20世纪生物学领域的一项新技术。FISH应用细胞遗传学和分子生物学的基本原理,作为架设细胞遗传学与分子生物学之间的桥梁,现已被广泛应用于植物学各方面的研究。本文就FISH的基本原理、技术发展及其在植物遗传育种、起源进化、染色体物理图谱构建方面的应用及发展趋势进行了综述。     

Fluorescence in situ hybridization (FISH)--application in research and diagnostics

The methods of molecular cytogenetics, in particular fluorescence in situ hybridization (FISH), are widely applied in cytogenetics for identification of numerical and structural chromosomal abnormalities, which are difficult to detect by routine cytogenetic techniques. Due to many advantages, FISH is used in research (gene mapping, gene expression studies, interspecies chromosome homology), and clinical diagnostics (chromosomal aberrations analysis in pre- and postnatal diagnostics, oncology). The techniques of in situ hybridization (ISH) are often employed in addition to classical banding techniques, in case where banding pattern is not reliable. This paper focuses on particular clinical examples, where FISH was successfully used to identify structural and numerical chromosomal aberrations.     

Heterogeneity of extraparenchymal primitive neuroectodermal tumors within the craniospinal axis

Four cases of primitive neuroectodermal tumors (PNETs) with unusual localization (three intraspinal extramedullary and one pontocerebellar) are reviewed. Histologically, they were small round blue cell tumors with diverse patterns. Immunohistochemically, all tumors were positive for at least two neuronal markers, two cases were Mic-2 positive and one showed glial differentiation. The paraffin-embedded tumor specimens were examined by interphase FISH using dual-color probes specific for EWS, HER-2 and BCR loci. Molecular cytogenetic study revealed the presence of EWS rearrangement in two cases and the presence of i(17q) in one tumor. Three tumors exhibited 22 disomy and one was 22 polyploid. Extraparenchymal PNETs within craniospinal axis are heterogeneous from the clinical, histological, immunohistochemical and molecular point of view. These PNETs can be of a central or peripheral type. Multidisciplinary approach is of a basic importance in differential diagnosis of such cases.     

Large-scale isolation of human 1p36-specific P1 clones and their use for fluorescence in situ hybridization

A series of 80 microclone probes derived from the chromosomal region 1p36 was used to isolate corresponding clones from the ICRF human P1 library (see Francis et al., this issue). Hybridization screenings were performed using probe pools on high-density filter grids. A total of 87 P1 clones specific for 1p36 were isolated. This large-scale approach allowed a detailed evaluation of the complexity, quality, and utility of this library. The isolated P1 clones were used both for size determination by pulsed-field gel electrophoresis and as probes for fluorescence in situ hybridization (FISH) analysis. FISH of P1 clones is shown to be both easy and efficient to perform on metaphase chromosomes and interphase nuclei. This observation is expected to reveal new avenues for diagnosis of disease-related chromosomal changes. The use of P1 clones as a tool in clinical and tumor interphase cytogenetics is discussed and compared with FISH data of other long insert clones such as cosmids and YAC clones.     

Utility of Interphase FISH Panels for Routine Clinical Cytogenetic Evaluation of Chronic Lymphocytic Leukemia and Multiple Myeloma

Specific genetic abnormalities are of prognostic significance for patients with chronic lymphocytic leukemia (CLL) and multiple myeloma (MM); however, routine cytogenetic analysis usually provides normal results. We utilized two probe panels for interphase fluorescence in situ hybridization (FISH) studies to enhance the ability to detect genetic abnormalities in samples that were referred for routine cytogenetic studies for possible diagnoses of CLL or MM. The CLL panel consisted of probes for 11q22.3 (ATM gene), 13q14 (D13S319), the centromere of chromosome 12 (D12Z3) and 17p13.1 (P53 gene). The MM panel included probes for 14q32 (IgH gene) and/or t(11:14)(q13;q32) (BCL1/IgH), 13q14 (D13S319) and 17p13.1 (P53 gene). FISH detected clonal aberrations not identified by conventional cytogenetics in an additional 8 of 23 (35%) samples referred for possible CLL and 7 of 42 (17%) samples with possible MM. The prognostic significance of the aberrations identified ranged from favorable, to intermediate, to poor. Our studies indicate that many samples referred for routine cytogenetics testing for CLL and MM yield normal results for both conventional and FISH testing, likely due to lack of definitive diagnosis in a percentage of cases. However, FISH is more sensitive for the detection of clinically significant chromosome abnormalities and should be the testing methodology of choice for these disorders.     

Interphase cytogenetics and comparative genomic hybridization of human epithelial cancers and precursor lesions

 The accuracy of cytogenetic analyses of human solid cancers has improved enormously over the past decade by the introduction and refinement of DNA in situ hybridization (ISH) techniques. This methodology can be applied to cells in the interphase state, thereby making it an excellent tool for the delineation of chromosomal aberrations in solid tumors. The use of non-isotopic ISH to intact and disaggregated cancer specimens will be discussed, as well as comparative genomic hybridization (CGH) with tumor-derived DNAs. In this review we will focus on hybridocytochemical interphase approaches for the detection of chromosomal changes in frequently occurring human epithelial malignancies, e.g., breast, lung, and prostate carcinomas. We will further discuss the use of ISH procedures for the genetic analysis of precursor conditions leading to invasive carcinomas. Knowledge concerning these precancerous conditions is increasing, and its importance in cancer prevention has been recognized. Interphase cytogenetics by ISH, as well as CGH, with DNAs derived from microdissected, precancerous, dysplastic tissue areas will increase our understanding of these lesions, both at the investigative and diagnostic levels. Accepted: 27 June 1997     

荧光原位杂交技术及其在医学诊断上的应用

荧光原位杂交技术是近年来生物学领域发展起来的将经典的细胞遗传学与分子遗传学结合起来一项新技术。该技术具有广泛的应用潜力,在细胞生物学、分子生物学、医学等众多领域快速发展。本文介绍了荧光原位杂交技术的基本原理和操作方法,并对该技术目前的发展状况以其在医学诊断上的应用进行了阐述。     

Comparative study of MLPA-FISH to determine DNA copy number alterations in neuroblastic tumors

Neuroblastoma tumor cells show complex combinations of genetic aberrations, and to date many different methods have been used for their detection. To apply genome-wide techniques, such as Multiplex Ligation-dependent Probe Amplification (MLPA), in routine diagnosis their validation is appropriate and necessary. DNA copy number alterations in 129 cases of neuroblastic tumors were detected using MPLA, and the results validated by Fluorescence In Situ Hybridization (FISH) (MYCN gene, 1p36, 11q and 17q). Kappa index values showed very good concordance between the two techniques in detecting homogeneous MYCN amplification (1); 11q deletion (0.908) and 17q gain (0.922). The validation results showed that MLPA is a highly efficient technique for diagnosis based on the genetic aberrations in relevant regions in neuroblastoma, showing a high concordance with FISH.     

Interphase cytogenetics

Conclusion Interphase cytogenetics is still in its infancy but the information which it is capable of providing will lead to a greater understanding not only of the normal interphase nucleus but also of the genetic content of tumor cells and will facilitate antenatal diagnosis of some hereditary diseases. Application to tumors will provide the ability to correlate chromosome complement (and ultimately single gene content) with tissue morphology and clinical tumor behaviour, perhaps providing prognostic information. We anticipate that this approach will give clues to consistent genetic abnormalities within tumors which can unambiguously be assigned to malignant as opposed to the normal stromal cell content of the tumor.Special issue dedicated to Dr. Sidney Udenfriend