E D I T O R I A L

See referenced original article on pages 977–88,this issue.

The author thanks Dr. Roger Mark for reading thearticle. The support of the staff of the LifespanAcademic Medical Center Cytogenetics Laboratoryis also acknowledged.

Address for reprints: Hon Fong L. Mark, Ph.D.,Cytogenetics Laboratory, Lifespan Academic Med-ical Center, Rhode Island Hospital, APC Building11th floor, 593 Eddy Street, Providence, RI 02903.

Received March 31, 1998; accepted March 31,1998.

Interphase Cytogenetics forStudying Solid TumorsHon Fong L. Mark, Ph.D.

Lifespan Academic Medical Center Cytogenetics Laboratory and Brown University School ofMedicine, Providence, Rhode Island.

Conventional cytogenetic analysis is a powerful, established tech-nique that can provide a picture of the human genome at a

glance. Most laboratories use G-banding using trypsin and Giemsastain (GTG-banding) for conventional cytogenetic analysis. In a rou-tine cytogenetic study, a short term culture is either established in thepresence of a mitogen—such as phytohemagglutinin (PHA), where itis called a stimulated culture— or grown without such an agent, inwhich case it is called an unstimulated culture. The former is used forperipheral blood cultures to rule out constitutional abnormalities,whereas the latter is used for the study of neoplastic tissues. Long-term tissue cultures are usually established for solid tumor studies.Harvesting chromosomes for conventional cytogenetics1 is a ratherlengthy and tedious process. Colcemid, a derivative of colchicine, isusually used to block spindle fiber formation and arrest the chromo-somes in metaphase. This is followed by a hypotonic treatment tocause the cells to take up water and swell so that the chromosomeswill spread well when dropped onto glass slides at a later step. Afterthe hypotonic treatment step, the cell pellet is fixed with a fixativeconsisting of three parts methanol to one part glacial acetic acid. Afterrepeated rinsings, the cells are then dropped onto glass slides andairdried. Slides are aged for a variable amount of time, then bandedand stained according to one of the banding protocols. GTG-bandingseems to be the most popular method in the U.S.,2,3 probably becauseof its simplicity. Prior to the advent of modern-day imaging systems,photographs were taken of the best metaphase spreads, and thephotographs were enlarged and hand-cut to separate the images ofthe chromosomes for identification. Most cytogenetics laboratoriesnow own computer-assisted karyotyping systems. Karyotyping in-volves arranging the 46 chromosomes in the human genome accord-ing to shape, size, and banding patterns. Thus, conventional cytoge-netics is a labor-intensive process requiring highly trained personnel.In addition, conventional cytogenetics depends entirely on the avail-ability of high quality metaphases, thus excluding from analysis thevast majority of cells that are in interphase.4

Conventional Cytogenetics of Solid Tumors Has Been HamperedThe conventional cytogenetics of solid tumors has been hampered bya number of factors,5–9 making an already lengthy and tedious processeven more challenging. One is the difficulty of establishing a cellculture. Another is the susceptibility to culture contamination despite

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adherence to sterile techniques. The tumor tissue maygrow at a very slow rate. The culture sometimes has alow mitotic index, resulting in insufficient cells forkaryotyping. The banding of the metaphases is attimes suboptimal, with fuzzy chromosomes. Even if allthese factors could be overcome, the resultant met-aphases could all be normal, leading one to wonderwhether the tumor cells have been overgrown by nor-mal stromal cells. The resultant karyotype, on theother hand, could be extremely complex, with multi-ple numeric and structural abnormalities leading todifficulties in interpretation. The possibility that theabnormal karyotype could be an artifact of culturealso cannot be completely ruled out. Due to thesevarious factors, conventional cytogenetics of solid tu-mors has not kept pace with that of hematopoieticdisorders, and few tumors are well characterized froma conventional cytogenetic standpoint.

In Situ HybridizationIn situ hybridization with radionuclide-labeled probeshas been reported since the late 1960s.10 Autoradiog-raphy, however, requires long periods of exposure thatare not practical for most applications. BiotinylatedDNA probes and probes modified with other reportedmolecules were introduced in the 1980s.5,11–13,14 –16

These early colorimetric in situ hybridization assayswere performed with immunocytochemical stains,such as horseradish peroxidase, and hybridization wasdetected with a light microscope. Some laboratoriestoday still adhere to these systems because of prefer-ence, whereas others have little choice because offinancial constraints. With the advent of fluorescence-based methods, termed fluorescent in situ hybridiza-tion (FISH),17 many laboratories began to use thisprotocol for both clinical and research applications.Regardless of whether the in situ hybridization is flu-orescence-based, it has the advantage of making anal-ysis of nondividing cells possible. This capability ofperforming cytogenetic analysis on interphase cells,called interphase cytogenetics, has indeed revolution-ized the field of cytogenetics.

Interphase Cytogenetics Complements ConventionalCytogeneticsInterphase cytogenetic analysis can be performed us-ing a variety of DNA probes, the most popular ofwhich are alpha-satellite DNA probes.18 Alpha-satel-lite DNA sequences are organized as tandem repeatsof unique 171 base-pair sequences that are present inas many as 5000 copies. For interphase studies, a DNAprobe to the pericentromeric alpha-satellite DNA of aspecific chromosome is biochemically modified bynonisotopic methods, such as nick translation with

biotinylated dUTP, and then hybridized to cells usingroutine hybridization techniques. Because of the highcopy number of the target DNA, highly sensitive, albeitcumbersome, radioactive detection methods are notrequired, and rapid, simple-to-use, nonisotopic detec-tion methods can be employed. Commonly used forthe detection of a biotinylated probe are fluorescein-ated avidin and avidin conjugated with a detectorenzyme, such as alkaline phosphatase. Both producepunctate signals that can be recognized microscopi-cally. The advent of digital imaging microscopy hasbrought further improvements in signal detection andsignal-to-noise ratios. The multiple applications of in-terphase cytogenetics have been discussed else-where.7,15

As already mentioned, the advantages of inter-phase cytogenetics include the fact that it is not re-stricted to cells arrested in metaphase, thus allowinganalysis of large numbers of cells. The ability to ana-lyze large numbers of cells is significant in that itpermits the detection of low frequency abnormalitiesthat are otherwise difficult to detect.4 Thus, specimensthat would not otherwise be suitable for analysis byconventional techniques could still yield useful cyto-genetic information. Interphase analysis could thus beused to study cells from tumors with low proliferativeactivity or from tumors that are difficult to maintain inshort term cultures. In addition, interphase cytogenet-ics is rapid because it does not require special trainingfor interpretation. Interphase cytogenetics allowssome correlation of cytogenetic findings with mor-phology, which is not possible with conventional cy-togenetic study because all nuclear details are lost in ametaphase cell. When used as an adjunct, interphasecytogenetics offers benefits in both the study of ma-lignant cells and the management of patients withmalignant disorders. It is ideal for archival tumor tis-sues that are amenable to immunocytohistochemicalstudies but not amenable to conventional cytogeneticanalysis.

For the above reasons and possibly others, inter-phase cytogenetics has been used to examine solidtumors, such as bladder tumors,19 –21 gestational tro-phoblastic disease,22 breast carcinoma,9,23–26 neuroec-todermal tumors,27 carcinoma of the testis,28 prostatecarcinoma,29 –31 and rhabdomyosarcoma.32

Interphase Cytogenetics Is Ideal for Studying Aneuploidy,a Potential Cancer BiomarkerAneuploidy has been explored as a biomarker forstratifying many cancers. Traditionally, flow cytom-etry has been the method of choice for ploidy analysis.Many studies suggest that DNA ploidy analysis usingflow cytometry and static image cytometry can pro-

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vide independent prognostic information in additionto stage and histologic grade.33,34 However, flow cy-tometry for detecting aneuploidy has its own limita-tions regarding sensitivity.35–37 For example, Visakorpiet al.36 found that FISH using three selected chromo-somes specific probes was two to three times moresensitive than cytometric DNA content analysis in an-euploidy detection. DNA flow cytometry, especiallywhen performed on paraffin embedded tumors, can-not distinguish aneuploid cell clones that have only afew numeric chromosomal changes from diploidclones. Early stage tumors that are often nearly diploidor show balanced chromosomal abnormalities are notdetected as DNA abnormal. Mesker et al.,38 for exam-ple, noted that the diagnostic value of DNA ploidymeasurements by flow or image cytometry with re-spect to the detection of DNA aneuploidy is limited,because only relatively significant changes in totalDNA content can be detected (i.e., at best 2%, depend-ing on the accuracy of the measurements). Generally,the equivalent of a gain or loss of one large or severalsmall chromosomes (a 4% change in total DNA con-tent) is required for adequate detection by flow cytom-etry.25,39,40 Thus, although the ability to evaluate largenumbers of nuclei readily is a strength of flow cytom-etry, a small population of abnormal cells may bedifficult to detect within a much larger population ofnormal cells.

In short, interphase cytogenetics based on thehybridization of DNA probes on interphase cells hasthe potential to revolutionize the field of surgical pa-thology because of its increased sensitivity over flowcytometry and static image analysis in aneuploidy de-tection.35,41,42 Previous studies have found that poly-merase chain reaction (PCR) techniques may not be assensitive as FISH due to tumor heterogeneity.43

Interphase Cytogenetics for Studying Aneuploidy as aPrognostic Marker in Prostate Carcinoma Illustrates theUtility of This General ApproachProstate carcinoma is a common disease affectingAmerican men, causing 40,400 deaths each year.44 Thecause of prostate carcinoma is currently not known.No specific genetic alterations associated with pros-tate carcinoma have been established so far.45

To date, tests measuring serum prostate specificantigen (PSA) levels combined with digital rectal ex-aminations and transrectal ultrasound– guided biop-sies have been the most reliable form of early detec-tion. Unfortunately, there are no available markers topredict clinical outcome accurately.43

Current prognostic indicators for prostate carci-noma are limited to Gleason grade and clinical stag-ing. As 244,000 new cases are diagnosed each year, the

need for more accurate methods to assess the extentof an individual patient’s cancer and define prognosticsubgroups has become urgent.

The optimal treatment for patients with prostatecarcinoma remains controversial. The failure to strat-ify patients into prognostic subgroups has hamperedthe recognition of the efficiency of aggressive localtherapeutic measures in comparison with watch-and-wait strategies.

In this issue of Cancer, a clearly written articlebased on a well-designed and scientifically significantstudy is presented by Henke et al.46 The study wasconducted to evaluate the relative role of interphasecytogenetics with chromosome enumeration probesand of conventional pathologic characteristics in thepreoperative prediction of postoperative tumor classi-fication and the recurrence of elevated serum concen-trations of PSA.

In the study of Henke et al., interphase cytogenet-ics was used to determine the chromosome copynumber. Chromosome enumeration probes for chro-mosomes 7, 17, and X were selected based on thefrequency of numeric chromosomal abnormalitiesfound in the authors’ previous studies of prostate car-cinoma patients.47,48 Six-micron sections of core biop-sies from 75 patients with clinically localized adeno-carcinoma of the prostate were used. A postoperativeincrease in PSA was used as an endpoint and an indexof outcome. Kaplan–Meier analysis of results showedthat the PSA recurrence ($ 0.4 ng/mL) was more fre-quent and observed earlier in patients with detectedchromosomal aneusomies compared with those whohad eusomic and tetrasomic chromosome numbers(P , 0.0001). Cox regression analysis indicated thatinterphase cytogenetics was the most valuable inde-pendent factor in predicting PSA recurrences. The au-thors concluded that the detection of numeric chro-mosomal aberrations in preoperative core prostatebiopsies is an adverse prognostic sign that is impor-tant for predicting prognosis and for selecting theappropriate form of therapy.

As Henke et al. noted, no molecular marker todate meets the criteria of a clinically relevant prognos-tic factor as defined by the College of American Pa-thologists.49 A reliable and consistent biomarker, or apanel of biomarkers, capable of defining therapy se-lection among subgroups is urgently needed. Geneticgrading based on assays of chromosome aneuploidymay ultimately prove to be the marker of choice forstratifying patients with prostate carcinoma and othercancers, although the number of patients studied todate has been relatively small and their follow-up timehas been short. The results obtained thus far are nev-

Interphase Cytogenetics and Solid Tumors/Mark 841

ertheless encouraging and should be confirmed andextended.

CONCLUSIONSAs Henke et al.46 noted, cancer development and pro-gression are complex processes involving multiple ge-netic alterations. Whereas the mechanisms in colorec-tal carcinoma have been elucidated, details of thegenetic events that influence carcinogenesis and ma-lignant progression in prostate carcinoma and mostother cancers remain poorly understood. Interphasecytogenetics using FISH with chromosome enumera-tion probes is ideally suited for the study of cancerusing archival materials.

Recent advances in molecular technology havenow led to the development of newer techniques thatcombine the sensitivity and specificity of FISH withthe global screening ability of conventional cytogenet-ics. Notably, spectral karyotyping, or SKY,50 is anevolving molecular cytogenetic technique that permitsexamination of the entire genome in a single hybrid-ization. However, because techniques such as com-parative genomic hybridization, or CGH,51–52 and SKYrequire special instruments and trained personnel, theexact roles that these new emerging technologies willplay in the average clinical cytogenetic laboratory inthe current climate of managed care and cost contain-ment53 is yet to be determined. Meanwhile, interphasecytogenetics using commercially available chromo-some enumeration probes is increasingly being uti-lized for the identification of potential biomarkers,such as chromosome aneuploidy and oncogene am-plification, which are both considered manifestationsof genetic instability that play pivotal roles in thegenetics and progression of human cancer.

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