839 E D I T O R I A L Interphase Cytogenetics for Studying Solid Tumors Hon Fong L. Mark, Ph.D. Lifespan Academic Medical Center Cytogenetics Laboratory and Brown University School of Medicine, Providence, Rhode Island. C See referenced original article on pages 977– 88, this issue. The author thanks Dr. Roger Mark for reading the article. The support of the staff of the Lifespan Academic Medical Center Cytogenetics Laboratory is also acknowledged. Address for reprints: Hon Fong L. Mark, Ph.D., Cytogenetics Laboratory, Lifespan Academic Medical Center, Rhode Island Hospital, APC Building 11th floor, 593 Eddy Street, Providence, RI 02903. Received March 31, 1998; accepted March 31, 1998. © 1998 American Cancer Society onventional cytogenetic analysis is a powerful, established technique that can provide a picture of the human genome at a glance. Most laboratories use G-banding using trypsin and Giemsa stain (GTG-banding) for conventional cytogenetic analysis. In a routine cytogenetic study, a short term culture is either established in the presence of a mitogen—such as phytohemagglutinin (PHA), where it is called a stimulated culture— or grown without such an agent, in which case it is called an unstimulated culture. The former is used for peripheral blood cultures to rule out constitutional abnormalities, whereas the latter is used for the study of neoplastic tissues. Longterm tissue cultures are usually established for solid tumor studies. Harvesting chromosomes for conventional cytogenetics1 is a rather lengthy and tedious process. Colcemid, a derivative of colchicine, is usually used to block spindle fiber formation and arrest the chromosomes in metaphase. This is followed by a hypotonic treatment to cause the cells to take up water and swell so that the chromosomes will spread well when dropped onto glass slides at a later step. After the hypotonic treatment step, the cell pellet is fixed with a fixative consisting of three parts methanol to one part glacial acetic acid. After repeated rinsings, the cells are then dropped onto glass slides and airdried. Slides are aged for a variable amount of time, then banded and stained according to one of the banding protocols. GTG-banding seems to be the most popular method in the U.S.,2,3 probably because of its simplicity. Prior to the advent of modern-day imaging systems, photographs were taken of the best metaphase spreads, and the photographs were enlarged and hand-cut to separate the images of the chromosomes for identification. Most cytogenetics laboratories now own computer-assisted karyotyping systems. Karyotyping involves arranging the 46 chromosomes in the human genome according to shape, size, and banding patterns. Thus, conventional cytogenetics is a labor-intensive process requiring highly trained personnel. In addition, conventional cytogenetics depends entirely on the availability of high quality metaphases, thus excluding from analysis the vast majority of cells that are in interphase.4 Conventional Cytogenetics of Solid Tumors Has Been Hampered The conventional cytogenetics of solid tumors has been hampered by a number of factors,5–9 making an already lengthy and tedious process even more challenging. One is the difficulty of establishing a cell culture. Another is the susceptibility to culture contamination despite 840 CANCER September 1, 1998 / Volume 83 / Number 5 adherence to sterile techniques. The tumor tissue may grow at a very slow rate. The culture sometimes has a low mitotic index, resulting in insufficient cells for karyotyping. The banding of the metaphases is at times suboptimal, with fuzzy chromosomes. Even if all these factors could be overcome, the resultant metaphases could all be normal, leading one to wonder whether the tumor cells have been overgrown by normal stromal cells. The resultant karyotype, on the other hand, could be extremely complex, with multiple numeric and structural abnormalities leading to difficulties in interpretation. The possibility that the abnormal karyotype could be an artifact of culture also cannot be completely ruled out. Due to these various factors, conventional cytogenetics of solid tumors has not kept pace with that of hematopoietic disorders, and few tumors are well characterized from a conventional cytogenetic standpoint. In Situ Hybridization In situ hybridization with radionuclide-labeled probes has been reported since the late 1960s.10 Autoradiography, however, requires long periods of exposure that are not practical for most applications. Biotinylated DNA probes and probes modified with other reported molecules were introduced in the 1980s.5,11–13,14 –16 These early colorimetric in situ hybridization assays were performed with immunocytochemical stains, such as horseradish peroxidase, and hybridization was detected with a light microscope. Some laboratories today still adhere to these systems because of preference, whereas others have little choice because of financial constraints. With the advent of fluorescencebased methods, termed fluorescent in situ hybridization (FISH),17 many laboratories began to use this protocol for both clinical and research applications. Regardless of whether the in situ hybridization is fluorescence-based, it has the advantage of making analysis of nondividing cells possible. This capability of performing cytogenetic analysis on interphase cells, called interphase cytogenetics, has indeed revolutionized the field of cytogenetics. Interphase Cytogenetics Complements Conventional Cytogenetics Interphase cytogenetic analysis can be performed using a variety of DNA probes, the most popular of which are alpha-satellite DNA probes.18 Alpha-satellite DNA sequences are organized as tandem repeats of unique 171 base-pair sequences that are present in as many as 5000 copies. For interphase studies, a DNA probe to the pericentromeric alpha-satellite DNA of a specific chromosome is biochemically modified by nonisotopic methods, such as nick translation with biotinylated dUTP, and then hybridized to cells using routine hybridization techniques. Because of the high copy number of the target DNA, highly sensitive, albeit cumbersome, radioactive detection methods are not required, and rapid, simple-to-use, nonisotopic detection methods can be employed. Commonly used for the detection of a biotinylated probe are fluoresceinated avidin and avidin conjugated with a detector enzyme, such as alkaline phosphatase. Both produce punctate signals that can be recognized microscopically. The advent of digital imaging microscopy has brought further improvements in signal detection and signal-to-noise ratios. The multiple applications of interphase cytogenetics have been discussed elsewhere.7,15 As already mentioned, the advantages of interphase cytogenetics include the fact that it is not restricted to cells arrested in metaphase, thus allowing analysis of large numbers of cells. The ability to analyze large numbers of cells is significant in that it permits the detection of low frequency abnormalities that are otherwise difficult to detect.4 Thus, specimens that would not otherwise be suitable for analysis by conventional techniques could still yield useful cytogenetic information. Interphase analysis could thus be used to study cells from tumors with low proliferative activity or from tumors that are difficult to maintain in short term cultures. In addition, interphase cytogenetics is rapid because it does not require special training for interpretation. Interphase cytogenetics allows some correlation of cytogenetic findings with morphology, which is not possible with conventional cytogenetic study because all nuclear details are lost in a metaphase cell. When used as an adjunct, interphase cytogenetics offers benefits in both the study of malignant cells and the management of patients with malignant disorders. It is ideal for archival tumor tissues that are amenable to immunocytohistochemical studies but not amenable to conventional cytogenetic analysis. For the above reasons and possibly others, interphase cytogenetics has been used to examine solid tumors, such as bladder tumors,19 –21 gestational trophoblastic disease,22 breast carcinoma,9,23–26 neuroectodermal tumors,27 carcinoma of the testis,28 prostate carcinoma,29 –31 and rhabdomyosarcoma.32 Interphase Cytogenetics Is Ideal for Studying Aneuploidy, a Potential Cancer Biomarker Aneuploidy has been explored as a biomarker for stratifying many cancers. Traditionally, flow cytometry has been the method of choice for ploidy analysis. Many studies suggest that DNA ploidy analysis using flow cytometry and static image cytometry can pro- Interphase Cytogenetics and Solid Tumors/Mark vide independent prognostic information in addition to stage and histologic grade.33,34 However, flow cytometry for detecting aneuploidy has its own limitations regarding sensitivity.35–37 For example, Visakorpi et al.36 found that FISH using three selected chromosomes specific probes was two to three times more sensitive than cytometric DNA content analysis in aneuploidy detection. DNA flow cytometry, especially when performed on paraffin embedded tumors, cannot distinguish aneuploid cell clones that have only a few numeric chromosomal changes from diploid clones. Early stage tumors that are often nearly diploid or show balanced chromosomal abnormalities are not detected as DNA abnormal. Mesker et al.,38 for example, noted that the diagnostic value of DNA ploidy measurements by flow or image cytometry with respect to the detection of DNA aneuploidy is limited, because only relatively significant changes in total DNA content can be detected (i.e., at best 2%, depending on the accuracy of the measurements). Generally, the equivalent of a gain or loss of one large or several small chromosomes (a 4% change in total DNA content) is required for adequate detection by flow cytometry.25,39,40 Thus, although the ability to evaluate large numbers of nuclei readily is a strength of flow cytometry, a small population of abnormal cells may be difficult to detect within a much larger population of normal cells. In short, interphase cytogenetics based on the hybridization of DNA probes on interphase cells has the potential to revolutionize the field of surgical pathology because of its increased sensitivity over flow cytometry and static image analysis in aneuploidy detection.35,41,42 Previous studies have found that polymerase chain reaction (PCR) techniques may not be as sensitive as FISH due to tumor heterogeneity.43 Interphase Cytogenetics for Studying Aneuploidy as a Prognostic Marker in Prostate Carcinoma Illustrates the Utility of This General Approach Prostate carcinoma is a common disease affecting American men, causing 40,400 deaths each year.44 The cause of prostate carcinoma is currently not known. No specific genetic alterations associated with prostate carcinoma have been established so far.45 To date, tests measuring serum prostate specific antigen (PSA) levels combined with digital rectal examinations and transrectal ultrasound– guided biopsies have been the most reliable form of early detection. Unfortunately, there are no available markers to predict clinical outcome accurately.43 Current prognostic indicators for prostate carcinoma are limited to Gleason grade and clinical staging. As 244,000 new cases are diagnosed each year, the 841 need for more accurate methods to assess the extent of an individual patient’s cancer and define prognostic subgroups has become urgent. The optimal treatment for patients with prostate carcinoma remains controversial. The failure to stratify patients into prognostic subgroups has hampered the recognition of the efficiency of aggressive local therapeutic measures in comparison with watch-andwait strategies. In this issue of Cancer, a clearly written article based on a well-designed and scientifically significant study is presented by Henke et al.46 The study was conducted to evaluate the relative role of interphase cytogenetics with chromosome enumeration probes and of conventional pathologic characteristics in the preoperative prediction of postoperative tumor classification and the recurrence of elevated serum concentrations of PSA. In the study of Henke et al., interphase cytogenetics was used to determine the chromosome copy number. Chromosome enumeration probes for chromosomes 7, 17, and X were selected based on the frequency of numeric chromosomal abnormalities found in the authors’ previous studies of prostate carcinoma patients.47,48 Six-micron sections of core biopsies from 75 patients with clinically localized adenocarcinoma of the prostate were used. A postoperative increase in PSA was used as an endpoint and an index of outcome. Kaplan–Meier analysis of results showed that the PSA recurrence ($ 0.4 ng/mL) was more frequent and observed earlier in patients with detected chromosomal aneusomies compared with those who had eusomic and tetrasomic chromosome numbers (P , 0.0001). Cox regression analysis indicated that interphase cytogenetics was the most valuable independent factor in predicting PSA recurrences. The authors concluded that the detection of numeric chromosomal aberrations in preoperative core prostate biopsies is an adverse prognostic sign that is important for predicting prognosis and for selecting the appropriate form of therapy. As Henke et al. noted, no molecular marker to date meets the criteria of a clinically relevant prognostic factor as defined by the College of American Pathologists.49 A reliable and consistent biomarker, or a panel of biomarkers, capable of defining therapy selection among subgroups is urgently needed. Genetic grading based on assays of chromosome aneuploidy may ultimately prove to be the marker of choice for stratifying patients with prostate carcinoma and other cancers, although the number of patients studied to date has been relatively small and their follow-up time has been short. The results obtained thus far are nev- 842 CANCER September 1, 1998 / Volume 83 / Number 5 ertheless encouraging and should be confirmed and extended. CONCLUSIONS As Henke et al.46 noted, cancer development and progression are complex processes involving multiple genetic alterations. Whereas the mechanisms in colorectal carcinoma have been elucidated, details of the genetic events that influence carcinogenesis and malignant progression in prostate carcinoma and most other cancers remain poorly understood. Interphase cytogenetics using FISH with chromosome enumeration probes is ideally suited for the study of cancer using archival materials. Recent advances in molecular technology have now led to the development of newer techniques that combine the sensitivity and specificity of FISH with the global screening ability of conventional cytogenetics. Notably, spectral karyotyping, or SKY,50 is an evolving molecular cytogenetic technique that permits examination of the entire genome in a single hybridization. However, because techniques such as comparative genomic hybridization, or CGH,51–52 and SKY require special instruments and trained personnel, the exact roles that these new emerging technologies will play in the average clinical cytogenetic laboratory in the current climate of managed care and cost containment53 is yet to be determined. 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