2011 Molecular Cytogenetics

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  • 96Review Article

    Molecular Cytogenetics: An Indispensable Tool for Cancer Diagnosis

    Thomas S. K. Wan, PhD; Edmond S. K. Ma1, MD

    Cytogenetic aberrations may escape detection or recogni-tion in traditional karyotyping. The past decade has seen anexplosion of methodological advances in molecular cytogenet-ics technology. These cytogenetics techniques add color to theblack and white world of conventional banding. Fluorescencein-situ hybridization (FISH) study has emerged as an indis-pensable tool for both basic and clinical research, as well asdiagnostics, in leukemia and cancers. FISH can be used toidentify chromosomal abnormalities through fluorescentlabeled DNA probes that target specific DNA sequences.Subsequently, FISH-based tests such as multicolor karyotyp-ing, comparative genomic hybridization (CGH) and arrayCGH have been used in emerging clinical applications as theyenable resolution of complex karyotypic aberrations and wholeglobal scanning of genomic imbalances. More recently, cross-species array CGH analysis has also been employed in cancer gene identification. The clini-cal impact of FISH is pivotal, especially in the diagnosis, prognosis and treatment decisionsfor hematological diseases, all of which facilitate the practice of personalized medicine.This review summarizes the methodology and current utilization of these FISH techniquesin unraveling chromosomal changes and highlights how the field is moving away from con-ventional methods towards molecular cytogenetics approaches. In addition, the potential ofthe more recently developed FISH tests in contributing information to genetic abnormalitiesis illustrated. (Chang Gung Med J 2012;35:96-110)

    Key words: molecular cytogenetics, fluorescence in-situ hybridization, multicolor karyotyping,comparative genomic hybridization, array CGH

    From the Department of Pathology, Queen Mary Hospital, University of Hong Kong, Hong Kong; 1Department of Pathology, HongKong Sanatorium & Hospital, Hong Kong.Received: June 14, 2011; Accepted: Oct. 25, 2011Correspondence to: Prof. Thomas S. K. Wan, Division of Haematology, Department of Pathology, Queen Mary Hospital, Universityof Hong Kong, Hong Kong. 102, Pokfulam Road, Hong Kong. Tel: 852-22553172; Fax: 852-28177565; E-mail: wantsk@hku.hk

    The rationale of classifying hematological malig-nancies is based on the separation of diseaseswith distinct clincopathologic and biologic features.Recognizing the association between specific cyto-genetic abnormalities and certain morphologic and

    clinical features, the World Health Organization hascategorized four unique subtypes of acute myelocyt-ic leukemia according to cytogenetics.(1) Therefore,cytogenetics study is currently considered a manda-tory investigation in newly diagnosed leukemia

    Prof. Thomas S. K. Wan

  • Chang Gung Med J Vol. 35 No. 2March-April 2012

    Thomas S. K. Wan and Edmond S. K. Ma FISH: an indispensable diagnostic tool


    owing to its usefulness in disease diagnosis, classifi-cation and prognostication. The vast majority ofrecurrent chromosomal rearrangements associatedwith leukemia were originally identified by cytoge-netic analysis, which remains the gold standard labo-ratory test since it provides a global analysis forabnormality on the entire genome. Although bandingtechniques represent the central theme at every cyto-genetics laboratory, it is sometimes difficult to kary-otype the tumor cells from a patient owing to unfa-vorable factors such as low specimen cell yield, lowmitotic index, poor quality metaphases and othertechnical difficulties. In addition, these techniquesdemand expertise such that the interpretation of vari-ant translocations or complex karyotypic configura-tions may challenge even the most experienced cyto-geneticist. The fluorescence in-situ hybridization(FISH) technique can be used to map loci on specificchromosomes, detect both structural chromosomalrearrangements and numerical chromosomal abnor-malities, and reveal cryptic abnormalities such assmall deletions. It has managed to overcome many ofthe drawbacks of traditional cytogenetics. FISH isroutinely applied in the clinical laboratory andallows nearly unlimited and targeted visualization ofgenomic DNA using either metaphase spread, inter-phase nuclei, tissue sections, or living cells. FISHapplications are particularly important for the detec-tion of structural rearrangements such as transloca-tions, inversions, insertions, and microdeletions, aswell as for identification of marker chromosomesand characterization of chromosome breakpoints.FISH is essentially a molecular technique which hasgreatly enhanced the accuracy and efficiency of cyto-genetic analysis by bringing together cytogeneticsand molecular biology. The impetus for many ofthese FISH technology innovations has been thedirect result of an increased understanding of thesequence, structure and function of the humangenome, which has highlighted the intricate marvelof the DNA architectural blueprint housed within ourchromosomes.(2,3) This review will summarize thedevelopment, current utilization and technical pitfallsof molecular cytogenetics techniques in clinical andresearch laboratories. Furthermore, this article high-lights how, with advancements in technology, thestudy of chromosomal abnormalities is moving awayfrom conventional methodologies towards molecularcytogenetics approaches.

    Use of FISH probes in the clinical laboratoryThere are a large number of good quality, direct-

    ly labeled commercial FISH probes available, ren-dering the technology accessible to clinical laborato-ries. They also provide strong signal intensity withlow background. The advantage of direct labeling forin-situ hybridization is that more than one probe maybe used simultaneously with each labeled with a dif-ferent fluorochrome. In the clinical laboratory, themost useful FISH probe systems are 1) centromericprobes, 2) chromosome painting probes, and 3) locusspecific probes for gene fusion, gene deletion orduplication.

    Centromeric enumeration probes (CEP)hybridize to the alpha (or beta) satellite repeatsequences within the centromeric regions specific foreach chromosome and are used for chromosomalenumeration. CEPs are applicable in demonstrationof trisomy, monosomy and ploidy level abnormali-ties. Chromosome painting probes are generatedfrom chromosome-specific probe libraries. They aredesigned to mark the entire chromosome of interest(Fig. 1A), and are useful in deciphering cytogeneticaberrations that are difficult to resolve on morpho-logical grounds, such as marker chromosomes ofuncertain nature or complex changes.(4) However,small or cryptic rearrangements of < 2-3 megabases(Mb) will not be uncovered using these probes.Locus specific probes hybridize to a unique sequencesite in the human genome. They are most frequentlyused to target genes of interest in order to detectrearrangements, gains, and deletions as well asamplification in both metaphase and interphase cells(Fig. 1A). Interphase analysis with FISH probes isquite an attractive and practical way to assess ampli-fication of v-erb-b2, erythroblastic leukemia viraloncogene homologue 2 (HER2) in human breast can-cer tissue sections, which identifies patients whomight benefit from trastuzumab (Herceptin) treat-ment (Fig. 2A).(5) In practical terms, FISH is consid-ered the best approach for detection of v-myc, mye-locytomatosis viral related oncogene, neuroblastomaderived (MYCN) amplification in childhood neurob-lastoma (Fig. 2B). It can distinguish between bonafide low levels of the MYCN amplification fromchromosome polysomy, and copy number hetero-geneity among tumor cells can be identified.(6)

    Interestingly, genetic heterogeneity in neuroblastomacan occur between primary tumor and bone marrow

  • Chang Gung Med J Vol. 35 No. 2March-April 2012

    Thomas S. K. Wan and Edmond S. K. Ma FISH: an indispensable diagnostic tool


    metastasis, and has been documented by FISH analy-sis.(6)

    There are two main systems of locus specificFISH probes for the detection of gene rearrange-ments.

    Dual color translocation probes

    The initial design of dual color translocationprobes in detecting chromosomal translocationsemploys the dual color single fusion system (S-FISH).(7) Typically, a probe labeled with one fluo-rochrome spans the 5end to the translocation break-point of a gene and another probe labeled with a dif-ferent fluorochrome spans the 3 end of the break-point of the partner gene (Fig. 2C). Thus, in ametaphase or an interphase harboring the transloca-tion, there is one signal each of the wild type allele

    and a fusion signal caused by juxtaposition of thefluorochromes as a result of gene fusion (Fig. 2D).However, the major drawback of the S-FISH systemis the relatively high false positive detection rateowing to close migration of two chromosomes oroverlap of signals by chance. This caveat is especial-ly important in the detection of low-level clones forminimal residual disease and in monitoring for earlydisease relapse. In order to tackle this drawback, thedual color signal fusion with extra signal (ES-FISH)system was subsequently developed.(7) The design isessentially the same as S-FISH but with a largerprobe spanning upstream and downstream of thetranslocation breakpoint of one of the two genesinvolved in the fusion, so that an extra signal (dimin-ished fluorescent intensity) is produced if the gene isdisrupted, in addition to signals of the wild type alle-

    A B

    C D E

    8 idup(8q)




    chromosome 8

    n = 13

    Fig.1 Cytogenetic characterization of a cancer cell line with various FISH-based approaches. (A) Metaphase FISH using a wholechromosome painting probe (green) and C-MYC probe (red), which shows tandem duplication of the C-MYC gene on both arms ofthe idup(8q) chromosome