Techniques in cancer cytogenetics: an overview and update.

Cancer Investigation, 10(2), 163-172 (1992)

TECHNOLOGY

Cancer Invest Downloaded from informahealthcare.com by McMaster University on 01/09/15 For personal use only.

Techniques in Cancer Cytogenetics: An Overview and Update Avery A. Sandberg, M.D., DSc. * and Julia A. Bridge, M.D.? The Cancer Center of Southwest Biomedical Research institute and Generrix 6401 East Thomas Road Scottsdale, Arizona 85251 ?Hattie B. Monroe Center for Human Genetics University of Nebraska Medical Center 600 S. 42nd Street Omaha, Nebraska 68198

In writing this minireview we have concentrated on methodological developments during the last decade and the last five years, in particular.

INTRODUCTION The correlation of nonrandom chromosomal abnormalities with specific types of human neoplasms is well established. Most of the karyotypic data available relate to the leukemias and lymphomas; however, recent advances in cytogenetic and molecular techniques have led to an increasing vat of genetic information concerning other neoplasias including both benign and malignant tumors. The present review focuses on the technical aspects of cytogenetic analysis of neoplasia and the interplay of several of the molecular genetic and cytogenetic techniques. An understanding of the technical advances in genetics and cytogenetics is of great importance because the results of these studies may influence the clinical management and bear upon the prognosis of patients with cancer and can be expected to ultimately lead to novel approaches with respect to prevention, diagnosis, and treatment.

SOLID TUMOR CYTOGENETICS The association of specific chromosomal abnormalities with particular clinicohistopathologic types of human neoplastic disease is well established. Much of the detailed information concerning significant chromosomal alterations is contained in several recent reviews (1-3). Karyotypic data regarding leukemias and lymphomas predominate, with solid tumors representing only about 15% of the total available data (3). The body of information of the karyotypic data of solid tumors is comparatively small when compared with that of the leukemias and lymphomas. A number of reasons exists: (a) many tumors, particularly the benign ones, have a low proliferative index, necessitating cell culture which 163

Copyright 0 1992 by Marcel Dekker, Inc.

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may be unsuccessful or may lead to overgrowth by nor-


mal s t r d cells such as fibroblasts; @) extensivenecrosis

or contamination by a coexistent infection may result in a low yield of sufficientmetaphases for analysis; (c) the morphology of the chranosomes in hematologic malignancies is often better for detailed analysis than in solid tumors; and (d) a host of chromsomal aberrations are frequently seen in solid tumors making it more difficult to discern the primary abnormality (43). In other words, it appears that solid tumors must overcome several levels of environmental controls and restrictions, making nonrandom cytogenetic changes more difficult to determine, since these tumors are likely to require more genetic steps than hematologic malignancies in the transformation and progression processes (6). Despite these obstaclesencounteredwith the karyotypic evaluation of solid tumors, refinemenis in cytogenetic techniques have led to an increasing number of reported studies.

Transportation of Tissues Tumor tissue may be transported in a small amount (5-10 ml) of sterile saline solution or phosphate-buffered saline (PBS)in a sterile contaimr suitable for transfer. For those specimens whose tramport requires a greater length of time (5-48 h), medium containing serum is preferable. RPMI-1640 medium supplementedwith 17 % fetal calf serum (FCS) and antibiotics (penicillin 100 unitdml, streptomycin 100 g/ml and gentamycin 50 g/ml) has often been used (7). L-15 medium has received recent attention as a particularly useful tmsport medium (8). Specimen samples may be kept at room temperature or refrigerated. If the tissue sample is large, it should be cut into smallerpieces before placing it in the sterile solution for transportation. On receiving the sample, if contamination is suspected, the tissue may be rinsed in sterilizing solution (culture medium and high concentrations of antibiotics) for 2 to 5 minutes (9).

Preparation of the Cultures and Culturing Four general techniques of processing cells for chromosome analysis are in present use: the direct method, suspension culture, mixed colony cultures, and in situ culture and harvest and techniques (10). Specimens are prepared for culture by disaggregationtechniques, which include mechanical and/or enzymatic processes. Enzymatic disaggregationpcedures with mllagenase II and DNase I are preferred over the mechanical method of mincing the tumor with scissors or scalpel or digestion

Sandberg and Bridge by trypsin (1 1,12). The former methods yield a larger number of viable cells and the quality of banding is superior. In addition, excellent results can frequently be obtained with long-term incubation with these enzymes (16 h to 6 days) (13). The direct method involves incubating the disaggregated tumor samples m culture medium containing colchicine (0.1-0.2 g/ml) for a few hours at 37 "C in order to collect metaphases from the in vivo dividing cells. The drawbacks of this method include an often insufficient quantity of metaphases and inferior banding quality (14). Suspension cultures involve incubation of the disaggregated tumor specimen in a serum-supplemented culture medium for 24-72 hours before harvest. The short-term suspension culture method has been applied to a number of different neoplasms but its success depends on the quantity of viable cells and their in vivo spontaneous mitotic activity. One of the most efficient methods of culturing is the mixed colony culture. With this method, a monolayer of cells is established on a polystyrene substrate and fed with culture medium supplemented with FCS and antibiotics (7). Chemical supplements, such as insulin and glutathione, can be added to the culture medium to help improve tumor growth, but these supplements are not essential (9). The cells are usually remved from the culture flask with an enzymatic treatment. Greatest success with this technique requires careful observation of the culture process with optimal estimation of the length and timing of colchicine exposure. Overgrowthof cultures by normal stromal fibroblasts that adhere more readily to plastic and are stimulated by high concentrations of serum is a problem frequently encountered with this method (15,16). The in situ method also involves the establishment of a monolayer of cells in culture, but the cells are normally grown and harvested in situ on a microscope slide or coverslip (10). The advantage of the in situ method includes conservationof technologist time and of cells that otherwise may have been lost during the centrifugation and pipetting steps involwd with standardharvesting techniques. A commercially available robotic harvesting system (modified Tecan Robotic Sample Processor Model 505) has been developed to further the efficiency of the in situ culture method. Reportedly, this system provides a consistent and accurate procedure that saves about 10-15 minutes of technologist time per case (10). Each of the above methods has its advantages and disadvantages. Selection of which technique to use depends on the intrinsic qualities of the tumor sample (i,e., sample size, degree of necrosis, and in vitro growth rate).


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Special Conditions

Needs in Solid tumor Cytogenetics

Since most human solid tumors require culturing of the affected cells for optimal cytogenetic analysis, methods leading to growth and division of these tumors have received much attention. For example, with the use of defined or partially defined media most human lung cancers can be successfully cultured (17)(Table 1). The culture retain the morphologic, biochemical, and cytogenetic properties of the tumors from which they were derived. Some tumors are more difficult to culture than others, with prostatic cancers being one of the most difficult. An improved technique for short-term culturing of human prostatic adenocarcinomaand its cytogeneticanalysis has been described (18).The method is based on: (a) prolonged mild collagenase treatment, (b) careful washing and repeated centrifugation and sedimentationof the disaggregated material to isolate viable prostatic epithelial cells, (c) short-term culture on collagen R-coated (Serva, Heidelberg, Germany) chamber slides with PFMR-4 medium (SBL,Stockholm, Sweden) supplemented with mitogenic factors, and (d) daily inspection of the cultured cells to determine the optimal time for harvesting. Another type of tumor difficult to culture and analyze cytogenetically is transitional cell carcinoma of the bladder. A method that allows routine cytogenetic analysis of small cytoscopic biopsies from urothelial tumors has been described (19).This method is based on prolonged mild collagenase treatment, a 12-16 h culture, and harvesting procedures adapted to give maximalmetaphase recovery. The use of phorbol-12,13-dibutyrateas a mitogen appears to be useful in the cytogenetic analysis of colon tumors (20).This compound alone or in combination with other agents (e.g., calcium ionophore A23187), may be applicable to many other tumors that are difficult to karyotype because of the inability to obtain mitoses.

Though a number of advances have been made in the cytogenetics of cancer and leukemia (Figs. 1-3),there is an array of areas that requires exploration and cogent methodologies. A partial list of these areas includes the following: 1. Stimulants for growth of cancer and leukemic cells, particularly the former. Failure of growth of affected cells occurs much too often in samples of carcinomas and epithelial tumors in general, in some leukemic states (e.g., chronic lymphocytic leukemia) and to a lesser extent in rnesenchymal neoplasms.

2. Spreading of the chromosomesin some metaphases is still far from optimal and new methodologies and/or preprations for swelling such cells are needed. 3. Though progress has been made in the recognition of the nature of the metaphase cells being examined cytogenetically,particularly in the marrow,a definite need exists for the similar recognition of tumor cells, especially in specimens in which a diploid picture is observed. 4. Approaches to improve the banding quality of leukemic and par-

ticularly cancer cells.

c

Table 1 Growth Factor Requirements for Culture of the Major Types of Lung Cancer All require insulin, transferrin, and a steroid hormone Non-SCLC require EGF Special requirements SLC: selenium, estradiol Adenocarcinoma: selenium, ethanolamine, albumin, T3 Squamous cell: s e m , cholera toxins, low Ca*+, feeder layers Source: From Ref. 17.

Figure 1. High-resolution chromosomes of a mrmal cell stained with Giemsa show various bands characterizing each chromosome. The aim of cancer cytogenetics is to develop methodologies which will yield metaphases of a similar nature. Unfortunately, this cannot be accomplished in a significant number of tumors, particularly carcinomas of various epithelial tissues.

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t 19

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& 21

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22

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Figure 2. A G - W e d kaqotypefrom a testicular germ cell tumcr showing the bands in the variau chromosome$ with some of the chromosomes approaching the quality in Fig. 1, but in others lacking such resolution, and, thus making their identification uncertain.

IN SITU HYBRIDIZATION In situ hybridization methodologies of metaphase chromosomes or interphase nuclei are techniques that allow the detection of specific DNA target sequences. The inchoation of these techniques occurred with such studies as those by Gall and Pllrdue (21) who used labeled 18 28s ribosomal RNA probes to detect genes in nucleoli of Xenopus Zuevis, and Jones (22) who localized mouse satellite DNA to the centric heterochromatic regions of mouse chromsomes. Localizationof the genes in these in situ hybridization studies was found with multiple gene copies, making detection easier because a large signal was generated by the many labeled probe mlecules within a small chromosome region. With recent technological advances, the sensitivity of the procedure has improved and now allows the detection of small u a u e gene sequences (low copy number) (23-26). A powerful techniquefor studying nudeic acid sequence organization and function in a wide variety of cell types and tissues is in situ hybridization of prophase or metaphase chromosomes. This approach has the capability

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of chromosomally mapping DNA sequences, directly detecting repositioning of sequences within the karotype as a result of chromosomal rearrangements, uncovering small rearrangements not detectable by standard karyotype analysis, and detecting and characterizing breakpoints using probes for defined DNA sequences (27-31). For example, in the case of the c-myb oncogene, in situ hybridization was carried out to sublocalize the gene, previously assigned to chromosome 6 (32). Hybridization with a 3H probe resulted in significant label at 6q2 with sublocalization of c-myb to 6q22-24 (33). These analyses are useful in further evaluating the involvement of this oncogene in malignanciesthat frequently involve 6q,such as acute lymphocytic leukemia (34), ovarian carcinoma (33, and malignant melanoma (36). The application of in situ hybridization methods for detecting and characterizing chromosomal breakpoints is well illustrated by the breakpoint on chromosome 22 in the variant t(8;22) in Burkitt lymphoma interrupting the lambda chain variable region genes (30) and the methallothionein gene cluster split by chromosome 16 rearrangements in acute myelomonocyticleukemia (31).


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MARKERS

Figure 3. G-baded karyotype of a synovial sarcoma with the t(X;18)@11;qll) characteristic of this tumor. A number of other cytogenetic changes are also present. The banding resolution achieved in this case is quite good, though in some chromosomes it is not optimal.

The in situ hybridizationtechnique allows direct examination of the position of my given cellular sequence in normal and transformed cells and may reveal translocations not detected by standard chromosomal analysis. In situ hybridization of interphase nuclei also allows detection of chromosomal aberrations. Numerical changes, deletions, and rearrangemenls of chromosoms can be rapidly delineated with this method. In addition, this method has the advantage of analysis of t u m r cells harvested directly from the primary cancer, without any cultivation step (37). Thus, this technique overcomes one of the major limitations of standard cytogenetic analysis, that of waiting and hoping for the recovered tumor cells to grow in culture and to contain analyzable metaphases (38). Furthermore, interphase in situ hybridization has provided valuable information concerning individual

interphase chromosome domains. The conclusions drawn by Manuelidis (39) in a study of interphase chromosomes by an in situ hybridization included the follotving: (a) human chromosomes can have a defined spatial position in the nucleus; (b) chromosomes in interphasecan be more extended than their metaphase counterparts; (c) despite unwinding, each human chromosome occupies a defined and localized nuclear domain; (d) each chromosome may have a characteristic three-dimensional configuration; and (e) 0.2-m wide interphase chromosome fibers are apparent. In situ hybridization of metaphase chromosomes or interphase nuclei can be achieved with radioactive or nonradioactive probes. In fact, methods for visualizing three nucleic acid sequences simultaneously utilizing and combining different fluorescent labels have been

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described (40). With in situ hybridization methods, the labeled probe is incubated under proper experimental conditions with the specimen to be examined. The probe is constructed to be complementary to the DNA being studied. These techniques rely on the availability of molecular probes for genes likely to be of clinical significance. Recently, a large array of probes has emerged through the use of recombinant DNA technology to identify and clone specific regions of DNA known to encode proteins of particular interest (41)Utilizing a bicrinylated probe specific for the lq12 band, lq polysomy was established in interphase nuclei of human solid tumors (breast and colon cancers) (42). Since such trisomy is one of the commonest changes in human neoplasia, this approach should prove to be useful in many ways. In summary, in situ hybridization serves as a powerful tool in elucidating fundmntal cellular events underlying the various pathologic characteristicsof cancer, and may be particularly useful when conventionalcytogenetictechniques are unrewarding.

CELC SYNCHRONIZATION Cell culture synchronizationis a method frquently used in combination with standard techniques. Cell culture synchronization can result in significantimprovement in the number of high-quality metaphases (hgh-resolution banding) obtained from stimulated lymphocyte preparations and leukemia cultures (43-47). Its utilization for the latter is controversial. For example, the efficacy of methotrexate (MTX) synchronization was assessed in marrow cultures from patients with ANLL and MDS (48). In contrast to cultures of stimulated lymphocytes from normal subjects, no improvement in mitotic index or metaphase quality could be detected. These authors also suggested that the use of intercalating agents may lead to more consistent improvements. The general opinion, however, holds that synchronization of cells in culture should increase the number of metaphases suitable for cytogeneticanalysis. The method most widely used to-date is that of MTX block and thymidine release (49). An advantage of synchronizedcell culture is the ability to obtain an increasednumber of divisions without extended exposure to colcemid, thereby maximizing the number of cells seen in the early stages of metaphase when the chromosomes are still long. MTX has been the most extensively employed synchronizing agent; it acts by reducing the levels of folic acid available to cells during DNA synthesis thus impairing de novo

purine and pyrimidine metabolism and leading to a block in nucleic acid synthesis. For optimal analysis, a sequential G- to R-banding protocol has been devised for high-resolution chromosomes (50). Attempts at utilizing hypoxic conditions for the growth of leukemic and cancer cells have, at least in our hands, not shown any efficacy that was preferential to the standard conditions of incubation or culture.

AUTOMATED CHROMOSOME ANALYSIS SYSTEMS One of the most recent advances in cytogenetic technology has been the development of fully or partially automated chromosome analysis systems. A number of different systems are commercially available, a few of which are listed in Table 2. The analytical stages of chromosome preparation normally involve metaphase scanning, chromosome counting, chromosome analysis, and production of karyotypes. The automated systems have been designed to improve both time and costeffectiveness of these procedures. All of the systems available are interactive and several offer automated metaphase finding as well as karyotyping. Finnon et al. (51) assessed the metaphase finding capability of one of the systems, the Cytoscana marketed by Image Recognition Systems (52). This model uses linear array cameras which enable very high speed scanning to be carried out (3-5 minhlide). Systematic metaphase finding can save time by scanning overnight and on weekends. Scanning stages are available that hold 1,4,8, or 16 slides. The same methods of routine preparation are used. An automated metaphase finder is particularly useful for those specimens in which the mitotic index is not very high and the metaphase

Table 2 Automated Chromosome Analysis Systems

Manufacturer Karyotec 100 Leitz MIAMED IK KATIE Genetiscan Cytoscan RK Sources: Refs. 51-55.

System Amcor Electronics, Ltd. E. Leitz, Inc.

General Imaging Corp. Perceptive Systems, Inc. Image Recognition Systems



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spreads are distributed over a wide area. The automated metaphase finder reduces tedium of exended searching (53). The systems are also useful when a large number of cells have be s c r e e d (i.e., fragile X). Automated chromosome analysis systems can achieve higher productivity. In particular, these systems have the advantage of eliminating darkroom time and costs, and the manual cutting and pasting of karyotypes. One of the biggest drawbacks of these systens, however, is the difficulty in obtaining high-quality images, a deficiency which has plagued the cytogenetics automation field since the 1960s. Nevertheless, new enhancement procedures have and continue to be developed in order to overcome this barrier (54). It is important to rcognize that these systems are primarily designed for quality banded amniotic fluid and peripheral blood specimens. Development of image analysis systems for neoplasms and plant chromosomes

is limited because of the presence of large chromosome numbers due to polyploidizationand difficulty in obtaining high-quality chromosome spreads, problems which are frequently encountered in these categories (55). Furthermore, the cost-effectivenessof such a system appears to be prohibitive at this time, to most cytogenetic laboratories.

MITOGENS AND GROWTH FACTORS Though some mitogens are available that are capable of stimulating specific cells, including a small number of malignant cells, e.g., &cell mitogens for the leukemic cells in chronic lymphocytic leukemia (CLL)(Table 3) and GCT for myeloid leukemic cells, a critical need exists for mitogens specific for various cancers, lymphomasand sarcomas. It is possible that some of the growth factors

Table 3 Mitogens for B and T Cells Mitogen

Amount

E. coli 055:B5 lipopolysaccharide Pokeweed mitogen Calcium ionophore A23187 Staphylococcal protein A (soluble) Stuphylococcur bacterial strain Cowan I protein (insoluble) Sodium metaperiodate (NaI04)

s

Source

0.1 ng/ml

Sigma, St Louis, MO

0.002 mglml

Sigma, St. Louis, MO

x 10-7 to 106 M 0.5-2.0 pg/ml 20-100 pglml 100 pglml 2to4

X

Calbiochan, San Diego, CA

1dM

T-cell growth factor

10% (vlv)

Cellular Products, Buffalo, NY

B-cell growth factors (20 kD)a

20% (v/v)

Cellular Products, Buffalo, NY

Conditioned medium for PHA-stimulated T cells (PHA-induced soluble factors)

30% (vlv)

Phytohemagglutinin (purified)

0.09 mglml

Burroughs Wellcome Co., Wellcome Reagents Div. Greenville, NC

20 pllml


Leukoagglutinin

10 pglml


Cytochalasin B

0. 05 pglml


Concanavalin A


Phorbol 12-myristate-13-acetate EB virus supernatant

10-20% supernatant; 1:9 v/v of culture

Interleukin-2 (IL-2)*

4 u/ml

Biogen, Geneva, Switzerland

Interleukin-7 (IL-7) @re-B- and T-cells)

30 U/ml

Immunex, Seattle, WA

Tells may require costimulation with anti-p Ab.

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(both natural and synthetic) may prove useful in that regard, though to date they have not been tried extensively in various conditions. It is also possible that growth inhibitors of some cells (e.g., fibroblasts) may also be useful in suppressing the growth of such cells so that the less mitotically active tumor cells have a chance to grow in vitro; a good example is cancer of the prostate in which the normal stromal cells tend to overgrow the cancer cells. It is apparent that much remains to be explored in establishing factors with inhibitor or suppressor activity in the cytogenetic examination of tumor cells.

LEUKEMIA CYTOGENETICS Results utilizing various preparations with possible stimulating activity of leukemic cells have not been consistent. Bone marrow leukemic cells of a variety of myeloid leukemias showed a pronounced increase in the mitotic index (5-50-fold) as compared to unstimulated cultures, and a greater than 100-foldincrease as compared to fresh, uncultured marrow cells in the presence of a conditioned medium derived from a human bladder carcinoma cell line (56). The addition of colony-stimulating factor (CSF) to cultures of leukemic myeloid cells, though not increasing the mitotic index significantly(1.5 times), caused more mitotic cells to be at the same stage (early metaphase or mid-metaphase) than seen in ordinary cultures (57). Similar observations were obtained with the use of conditioned medium from a culture of a giant cell tumor (58,59), Conditioned media from a human lung adenocarcinoma cell line expressing interleukins 1 and 6 (IL-1 and IL-6) and granulocyte (G), macrophage (M) and GM colonystimulating factors (G, M, and GM-CSF) failed to stimulate growth of bone marrow cells from patients with leukemia, myelodysplastic syndrome or lymphoma. In fact, the results indicated that the conditions favored growth of normal cells versus leukemic cells and thus may mask cytogenetic abnormalities (60). The sequential addition of fluorodeoxyuridineand then thymidine to cell colonies (Ph from CML) growing in semisolid medium yielded higher mitotic rates by the synchronization of cells. This method also improved the general quality of the chromosome preparations. The authors (47) indicated that minor changes in this method should make it useful for the analysis of colonies derived

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from a variety of other normal or malignant clonogenic cell types. Controversy continues to exist regarding the efficacy of direct examination of leukemic marrows versus 24-h cultures versus mitotic synchronization. In one study (61) no apparent differences were found in the karyotypes of 90 patients with hematologic disorders among the three methods. Also, the differences in quality or number of metaphases found among the three methods were not statistically significant, though 24-h unstimulated cultures produced more metaphases than the synchronization procedure. The authors suggested that in routine practice at least two different methods be used and it may be best if at least one of these methods is a direct technique. A reliable way for optimal G-banding of leukemic metaphases involves the incubation of slides immediately following air-drying for 90 minutes in an oven (62). Inside the oven, the temperature should be 100-110°C and a humidity of 98% (heated by placing several beakers of distilled water in the oven). Hot slides are stained immediately after removal from the oven. The techniques for obtaining karotypic findings in the chronic and acute leukemias have been steadily improved over the years (1). Factors which play a role in this success, particularly in ALL, include sample type (marrow vs. blood cells), sample cell count (concentrationof blasts and total white blood cell count), and time of transit (less than 24 h) (63,64). Generally, the highest success rate is seen in Ph+ CML, followed by ANLL, MDS, and then ALL. In recent years techniques have been developed that allow the recognition of the type of cell on which the karyotype has been established, in particular the cells of marrow origin. Several approaches have been taken to determine the nature of the cells studied cytogenetically (65)

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1. Cytogenetic studies of single colonies from stem cell cultures (66)

2. Concurrent and comparative analysis of separatedpreparations from the same sample (67) 3. Cytogenetic analysis of cells that have been sorted according to their phenotype with a cell sorter (68) 4. Simultaneousdeterminationof both karyotype and phenotypeof the same cell (69-72)

The first three methods provide only indirect evidence for the origin of metaphases, whereas the fourth permits a direct phenotypic and cytogenetic correlation at an individual cell level (72).


THE CANCER CYTOGENETIC1SI"S DREAM


Under ideal conditiom the cells whase chromosome constitution we wish to know would divide preferentially in vitro to other cells and automated instrumentationwould identify the nature of the cells, tell us the number of chromosomes in perfectly spread preparations, indicate the cytogenetic anomalies present and the exact chromosomes and their bands affected, signify the possible gene involved or affected, prepare a karyotype of the cell and point to the recurrent or specific karyotypic changes present in the ell(s).

The studies referred to in this presentation a d originating in the authors' laboratories have been supported, in part,by Grants CA-41183 and CA-14555 from the Natbnal Cancer Institute and the Orthopaedic Research Education Foundation.

Address reprint requeststo Awry A. Sandberg, MD., D.Sc., The Cancer Center of Genetrix, 6401 East Thomas bad, Scottsdale, A2 85251.

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Cancer and thrombosis--an update.

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minimisation.