Comparison of cytogenetics, interphase cytogenetics, and DNA flow cytometry in bone tumors

Cytometry (Communications in Clinical Cytometry) 26:185-191 (1996)

Comparison of Cytogenetics, Interphase Cytogenetics, and DNA Flow Cytometry in Bone Tumors

Maija Tarkkanen, Stig Nordling, Tom Bohling, Aarne Kivioja, Erkki Karaharju, Jadwiga Szymanska, Inkeri Elomaa, and Sakari Knuutila

Departments of Medical Genetics (M.T., J.S., S.K) and Pathology (S.N., T.B.), Haartman Institute, University of Helsinki, and Departments of Orthopaedics and Traumatology (A.K., E.K) and Oncology (I.E.), Helsinki University Central Hospital,

Helsinki, Finland

Twenty-three samples of benign and malignant bone tumors were studied with cytogenetic analysis, interphase cytogenetics (IC) using in situ hybridization with (peri)centromeric probes for chromosomes 1, 7, and/or 8, and DNA flow cytometry (FCM). Our aim was to compare these methods in the detection of numerical chromosome aberrations (NCA) and aneuploidy. IC detected aneuploidy in 91%, FCM in 73%, and cytogenetics in 27% of the malignant tumors. In benign tumors IC detected aneuploidy in 4 (33%), FCM in 2 (17%), and cytogenetic analysis in 1. Al l of the benign tumors aneuploid by IC, two of which were also aneuploid by FCM, were histologically potentially aggressive. The clonal aberrations detected with cytogenetics usually agreed with the IC and FCM findings. Al l malignant tumors which had a normal karyotype were aneuploid either by IC or FCM or by both. In conclusion, IC was the most sensitive method in the detection of NCA and aneuploidy even though it was usually performed with only two (peri)centromeric probes. Aneuploidy was detected by cytogenetic analysis alone in 4 samples (1 7%), by cytogenetic analysis and/or FCM in 1 1 samples (a%), and by cytogenetic analysis, FCM, and/or IC in 16 samples (70%). Thus, the combined use of all three methods increased the sensitivity of aneuploidy detection. 0 1996 Wiley-Liss, Inc.

Key terms: Cytogenetic analysis, interphase cytogenetics, in situ hybridization, DNA flow cytometry, numerical chromosome aberrations, ploidy, bone tumors

Few diagnostic genomic markers are known for bone tumors and their therapeutic and prognostic applications are still very limited. Genomic alterations of a tumor can be studied with several methods. In cytogenetic analysis, approximately 20-30 metaphases are examined. It gives an overview of the chromosomal changes and can reveal very small structural aberrations. However, cell culture can work as a selective force by selecting a subclone, perhaps only a minor clone in vivo, but maybe the only clone succeeding in vitro. In bone tumors metaphases are difficult to obtain and their quality is often poor. A normal karyotype can reflect stromal, non-neoplastic cells; on the other hand, the tumorigenic changes could be detectable only at the molecular level.

In interphase cytogenetics (IC), in situ hybridization (ISH) of chromosome-specific probes detects numerical chromosome aberrations ( N U ) ( 1 1,22,29). Usually at least 100 cells are analyzed. The analysis does not require mitotic cells and can be performed on histologic sections of paraffin-embedded tissue. One disadvantage of IC is that only small regions, usually the (peri)centromeric areas, can be recognized by the probes. The number of probes which can be used is often limited.

0 1996 Wiley-Liss, Inc.

DNA flow cytometry (FCM) is a fast method, giving an estimation of the DNA content after analyzing usually more than 10,000 cells. One disadvantage of FCM is that it only measures the total amount of DNA; therefore, bal- anced aberrations, e.g., translocations, are not detected. In addition, FCM will only detect differences in the DNA content greater than 4% (23). Thus, while cytogenetics and IC can reveal alterations affecting only one chromosome or a chromosomal region, the smallest change de-

Received for publication October 25, 1995; accepted January 18, 1996.

This work was supported by a grant from Zeneca Pharma to the Foun- dation for the Finnish Cancer Institute (M.T.), the Clinical Research Institute of the Helsinki University Central Hospital (M.T.), the Finnish Medical Society Duodecim (M.T.), Finska Wkares3llskapet (T.B.), the Finnish Academy of Sciences (I.E.), and the Finnish Cancer Society (S.K., M.T.).

Address reprint requests to Maija Tarkkanen, M.D., Haartman Insti- tute, Department of Medical Genetics, P.O. Box 2 1 (Haartmaninkatu 31, FIN-00014 University of Helsinki, Finland. E-mail: Maija.Tarkkanen @Helsinki. Fi.

186 TARKKANEN ET AL.

tectable by FCM corresponds approximately to a gain or loss of a whole chromosome 1 or 2 (25). A change in less than 5% of the cell population is usually missed. FCM provides no information about the individual chromosomes participating in the aberrations.

Previous studies on various cancers have compared cytogenetic analysis with FCM (14,24,31,33,39) or IC (30,32), and IC with FCM (2,3,8,9,20,21,28,34,41); few have combined all three methods (4,5,38). Simultaneous use of these methods has usually improved the sensitivity for the detection of aneuploidy and NCA. The results are often in agreement, but conflicting results have also been reported, possibly because of the heterogeneity of tumors and the varying sensitivity of the methods. In the present study we compared cytogenetic analysis, IC, and FCM in bone tumors in the detection of NCA and aneuploidy. To our knowledge, this is the first study utilizing all three methods simultaneously on bone tumor samples.

MATERIALS AND METHODS Material

Twenty-six samples from 24 patients were included (Table 1). The selection criteria were that a cytogenetic analysis, IC, and FCM had been performed on the same tumor. The material consisted of 12 benign (including 3 tumor-like lesions: fibrous dysplasia, aneurysmal bone cyst, and lipoma) and 9 malignant tumors, and 2 pulmonary metastases of osteosarcoma. Three non-neoplastic bone lesions were used as controls (nos. 1-3) for IC and FCM. Three patients had received chemotherapy before the operation. The tumors previously described in refs. (36) and (37) are indicated in Table 1.

Cytogenetics The method has been described earlier (36). Chromo-

some preparations were made after a mean of 9 days of culture and, when possible, at least 20 metaphases were analyzed. The karyotypes were determined according to ISCN 1991 (27). The chromosome index (CI) was determined by dividing the modal chromosome number by 46 (24). If a tumor had a normal karyotype with non-clonal aberrations, the modal number was assumed to be 46. In tumors with chromosome number variation, the mean of the CI interval was used. Separate CIS were determined for each subclone in a tumor.

IC by ISH Uncultured cell suspensions, obtained by mechanical

disintegration of the tumor tissue, were fixed in metha- no1:acetic acid (3:l) and screened for NCA. Since usually only a small amount of cell suspension was available from each tumor, only three probes were used: pUC1.77 (# 1) and pa7tl (# 7) recognize repeat sequences in the pericentromeric region of chromosome l (lq12) (10) and in the centromeric region of chromosome 7 (42). For chromosome 8 (# 8) a centromere-specific probe was gen- erated by polymerase chain reaction (PCR) with oligo- nucleotide primers for the conserved region of the

alpha satellite monomer (13) with a chromosome 8 li- brary probe as template (LLO8NSO2, American Type Cul- ture Collection, Rockville, MD). These chromosomes have been reported to be affected in bone tumors, especially in malignant tumors (6,19,26,36). Sixteen samples were studied with probe # 1 and # 7, one sample with # 1 and # 8 and one with # 7 and # 8, two samples with # 1, # 7, and # 8, and six with one of the probes.

The slides were pretreated with 0.01 N HCl, followed by pepsin (Sigma, St. Louis, MO) at 0.01 mg/ml in 0.01 N HC1 for 5-6 min, washed in distilled water, and dehy- drated in an ethanol series. Slides for # 1 and # 7 were further incubated in methanol containing hydrogen per- oxide followed by ethanol. In general, the hybridizations were carried out as described (29). In brief, the probes were labeled by nick translation (Nick Translation Kit, Bethesda Research Laboratories, Gaithersburg, MD): # 1 and # 7 with biotin- 1 1-dUTP (Sigma) and # 8 with digoxigenin-11-dUTP (Boehringer Mannheim GmbH, Ger- many). After denaturation the hybridization was carried out at 42°C overnight (# 1, # 7) or for 2 days (# 8).

Probes # 1 and # 7 were detected by incubating the slides in normal rabbit serum, in mouse antibiotin anti- body, followed by peroxidase-conjugated rabbit anti- mouse serum (all from Dakopatts A / S , Glostrup, Den- mark). The color was developed with diaminobenzidine tetrahydrochloride (Sigma), and the slides were counterstained with hematoxylin. Probe # 8 was detected with mouse anti-digoxigenin followed by sheep anti-mouse an- tibody conjugated with fluorescein isothiocyanate (all from Sigma). The slides were counterstained in an anti- fade solution with DAPI (Sigma).

Usually at least 100 cells were analyzed. The mean numbers of cells analyzed were 135, 127, and 174 for probe # 1, # 7, and # 8. Only intact, non-overlapping nuclei in which the signals were of uniform intensity and size and completely separated from each other were analyzed (22). Split signals, where the two components were smaller than the other signals in the same nucleus, were counted as one signal. Such signals arise from the pericentromeric repeat being divided by the centromere (17). In the non-neoplastic controls the number of cells with more than two signals never exceeded 3%. There- fore, and also based on previous IC studies on nuclear suspensions (4,7,8,12,28,30), a sample was designated as aneuploid if more than 5% of the cells contained three or more signals.

FCM Nuclear suspensions were prepared from 21 fresh tu-

mor samples as previously described (16). The DNA content of about 20,000 cells was measured with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) using an argon laser. The excitation wavelength was 488 nm, and the total emission above 580 nm was measured. HL-60, a human leukemia cell line known to have a diploid DNA content, served as an external control and chicken red blood cells (ChRFK) were added as an in-

CYTOGENETICS, IC, AND FCM IN BONE TUMORS 187

Table 1 Histopathologic Diagnoses and Karyotypes of 23 Specimens of Bone Tumors and Tumor-Like Lesions and the Results Obtained

From Cytogenetic Analysis, FCM, and ICe

Patientb Diagnosis' Karyotype CI DI IC aneuploidyd I t Control 46,XX 1.00 1.00 # 1 - # 7 - 2' Co ntro I 46,XY 1.00 1.00 # 1 - # 7 -

3' Control 46,XY 1.00 1.00 # 7 - 4 Fibrous dysplasia 46,XX 1 .oo 1.00 # 8 - 5 Aneurysmal cyst 46,XY 1.00 1.00 # 1 - # 7 - 6' Lipoma 46,XX 1.00 1.00 # 1 - # 7 - 7 Enchondroma 46,XX 1 .oo 1.00 # 1 - 8 Chondroma 46,XX 1.00 1.00 # 1 - # 7 -

9t Chondroblastoma 46,XX 1.00 1.00 # 1 + 10' Giant cell tumor grade II 46,XX,-6,-7,-7,-12, 1 .oo 1.00 # 1 - # 7 -

11' Giant cell tumor grade II 46,XY 1.00 1.00 # 1 - # 7 -

# 8 -

# 8 -

# 7 -

+ 2-5mar, inc[cp61/46,XX[91

12a Giant cell tumor grade II (R) 46,XX + non-clonal changes 1.00 1.05 # 7 + + # 8 + +

12b Giant cell tumor grade II (R) 46,XX + non-clonal changes 1.00 1.08 # 1 + + # 8 + + # 7 + +

14' Parosteal osteosarcoma (R) 46,XY + non-clonal changes 1.00 1.92 # 1 + + + # 7 + + +

15t Periosteal osteosarcoma 46,XY 1.00 1.36 # 1 + +

13a Giant cell tumor grade Ill 46,XX + non-clonal changes 1 .oo 1.00 # 1 + 13b Giant cell tumor grade Ill 46,XX 1.00 1.00 # 1 - # 7 -

grade II # 7 + + 16' Osteosarcoma grade IVt 46,XY 1 .oo 1.00 # 1 + +

18' Osteosarcoma, pulmonary 46,XX + non-clonal changes 1 .oo 1.00 # 1 + + metastasis grade IVt # 7 + +

l g t Osteosarcoma, pulmonary 46,XY 1.00 2.11 # 1 - metastasis grade I I I-IV

20' Chondrosarcoma grade I I I 46,XX 1.00 1.00 # 1 +

# 7 - 17t Periostea I osteosarcoma 46,XX 1.00 0.62 # 7 -

grade IVt

# 7 + + 21t Chondrosarcoma grade Ill 50-65,+marl, +mar2, 1.25 2.97 # 7 + + + 22t Chondrosarcoma 34-42,XY,?t( 1 ; lO)(p22;pl5), -2, i(6)(p10), 0.83/1.74 0.88 # 1 + +

+mar3, + mar4,inc[cp41

add(19)(~13), -22. + 1-3mar[c~41/ 77-83. grade Ill (R) i(9)(qlO),-ll, - 12, - 13,-14,- 17, # 7 +

idemx2, inc[cp21/46,XY[ll 23* Ded ifferentiated 46,X,-X,t(9;22)(q34;q 11-12),

chondrosarcoma grade IV + mar[51/52-61.?X. 1.00/1.23 3.34 # 1 -

# 7 + + + - 2-3 dm i n, i nc[91/46,XX[21

24' Fi brosarcoma grade I I I 46,XY 1.00 1.84 # 1 - # 7 + + aCI = chromosome index; DI = DNA index. bThe cases marked with ' have been previously published in ref. (36) and that with * in ref. (37). Samples 12a and 12b represent

the same patient with a recurring giant cell tumor. Samples 13a and 13b represent the same patient: 13a obtained from the biopsy and 13b from the resection.

'Samples marked with were taken after chemotherapy. R = tumor recurrence. dA sample was interpreted as aneuploid if more than 5% of the cells contained three or more signals. - = no aneuploidy, + =

6-10% of cells aneuploid; + + = 11-20%; + + + = >20%.

ternal control. The DNA index (DI) was calculated by dividing the mean channel of the GO/G1 peak of the sample by the mean channel of the GO/G1 peak of the diploid reference and correcting for the channel values of the ChRBC. Tumors were classified as aneuploid if there was at least one GO/G1 peak in addition to the diploid peak. If the first or only G1 peak had a DI between 0.9 and 1.1 the DI of this peak was given the value 1.0. Nuclear suspen-

sions were obtained from paraffin-embedded tissue in five tumors using a modification of the method described by Hedley et al. (15,18). In four tumors, only paraffin- embedded specimens were available. An unexpected result from the fresh specimen of one lesion was controlled using an archival specimen (# 4). For five tumors, FCM data from fresh samples were available from preceding or following operations (Table 2).


Table 2 Follow-Up of FCM Findings

Present study Time between Preceding Or following sample

4 Fibrous dysplasia Biopsy 1.00 + 154 Evacuation 1.17

14 Parosteal osteosarcoma Resect ion 1.92 -37 Biopsy 1.93 1 7a Periosteal osteosarcoma grade IV Resection 0.62 -73 Biopsy 0.61

12 Recurring giant cell tumor grade II Evacuation (12a) 1.05 + 80 Biopsy (12b) 1.08

Patient Diagnosis Type of operation DI operations (days) Type of operation D\

8 Chondroma Biopsy 1.00 + 5 1 Evacuation 1.00

20 Chondrosarcoma grade Ill Biopsy 1.00 + 57 Resection 1.00

13 Giant cell tumor grade Ill Biopsy (13a) 1.00 + 3 6 Resection (13b) 1 .oo aChemotherapy between the operations.

RESULTS Cytogenetics

One of the 12 benign tumors had a clonal aberration, 8 had normal karyotypes, and 3 had normal karyotypes with non-clonal aberrations (Table 1 ). Three of the 11 malignant tumors had clonal aberrations, 6 had normal karyotypes, and 2 had normal karyotypes with non-clonal aberrations.

IC by ISH There was no evidence of NCA in 9 of the 23 tumors

(39% ) 1 osteosarcoma operated after chemotherapy (# 17, Table 1) and 8 benign tumors. Thus, 8 of the 12 (67% ) benign tumors were diploid by IC (Table 3). Four- teen of the 23 tumors (61% ) were aneuploid by IC with at least one probe: 10 of the 11 malignant tumors (91%) and 4 of the 12 (33%) benign tumors (Table 3). These four tumors were a chondroblastoma (# 9), a grade 111 giant cell tumor (# 13a), and two samples of a recurring grade I1 giant cell tumor (# 12a and # 12b).

Comparison of IC Findings in Successive Samples Sample 12a was studied with probes # 7 and # 8 and

sample 12b with # 1 and # 8. The frequency of cells exhibiting more than two signals for probe # 8 was iden- tical in both samples (20% ). Both samples of tumor 13 were studied with probe # 1 and # 7. The frequencies of cells with more than two signals were 8% and 4% in samples 13a and 13b with probe # 1 and 15% and 1% with probe # 7, respectively.

FCM Thirteen tumors were diploid by FCM (57% ; Table 1 ),

10 benign and 3 malignant tumors: a grade I11 chondrosarcoma (# 20), a grade IV osteosarcoma (# 16), and a lung metastasis of an osteosarcoma (# 18) (16 and 18 obtained after chemotherapy). Ten samples were aneuploid by FCM (44% ), of which two were from the same recurring giant cell tumor (12a-b), and eight malignant.

Follow-Up of FCM Findings Additional FCM results were obtained for five patients

from operations preceding or following the ones of the present study (Table 2). The results agreed in four tu-

Table 3 Comparison of Results Obtained by Cytogenetic Analysis, IC, and DNA FCM in 23 Tumors and Tumor-Like Lesions of Bone

Malignant Benign tumors (%I Tumors (%I

Aneuoloid IC 91 DNA FCM 7 3 Cytogenetic analysis 27

33 17 8

Diploid IC 9 67 DNA FCM 27 8 3 Cvtoeenetic analvsis 7 3 9 2

mors and disagreed in one (# 4). The FCM results of successive samples from the tumors of two patients (# 12 and # 13) were similar (Tables 1, 2).

Tumors With Clonal Aberrations: Comparison of Cytogenetics, IC, and FCM

A grade I1 giant cell tumor (# 10) had a pseudodiploid karyotype with four chromosomes missing from the C group, including both homologues of chromosome 7, and with two to five clonal markers. These were of approximately the same size as C group chromosomes. The DI was 1.00. There was no evidence of gains of chromosome 1 or 7, neither was there any evidence of nullisomy for chromosome 7 in IC (data not shown). A grade 111 chondrosarcoma (# 21) had a hypotriploid karyotype with complex changes [chromosome number range (CNR) 50-65; CI 1.251. The DI was hexaploid (2.97). IC with probe # 7 was aneuploid. A grade I11 chondrosarcoma (# 22) had a hypodiploid stemline clone with multiple aberrations (CNR 34-42; CI 0.83) and a hypotetraploid sideline clone (CNR 77-83; CI 1.74). The DI was hypodiploid (0.88). IC was aneuploid with probes # 1 and # 7. A high-grade chondrosarcoma (# 23) had pseudodiploid and hypotriploid clones (CNR 52-6 1 ; CI 1.2 3). FCM of a paraffin-embedded specimen revealed a hyper- hexaploid clone (DI 3.34). The tumor has previously been reported to have a DI of 1.9 (37). This was based on a fresh sample with very few cells and, therefore, is prob- ably incorrect. IC was aneuploid with probe # 7 but not with probe # 1.


Table 4 Comparison of Ploidy Patterns by IC and FCM in 23

Tumors and Tumor-Like Lesions of Bone

IC ploidy DNA FCM ploidy Diploid Aneuploid

Diploid 8 5 Anemloid 1 9

DISCUSSION In the present study IC was the most sensitive method

to detect NCA and aneuploidy even though it was usually performed with only two (peri)centromeric probes. The combined use of the three methods increased the sensitivity of aneuploidy detection as aneuploidy was detected by cytogenetic analysis alone in 4 samples ( 17% ), by cytogenetic analysis and/or FCM in 1 1 samples (48% ), and by cytogenetic analysis, FCM, and/or IC in 16 samples (70% ).

Fourteen tumors were aneuploid by IC, 9 of which were aneuploid also by FCM (Table 4) , so the accuracy of FCM to detect aneuploidy detected by IC was 64%. The factors which could explain the discrepancies between IC and FCM are: First, as different tissue samples were analyzed by these methods, tumor heterogeneity may have caused some of the differences. Heterogeneity has been detected in mesenchymal tumors with cytogenetic analyses (43 ) . Second, the aberrations may have been too slight to be detected by FCM as it only detects changes greater than 4% in the DNA content (23 ) .

Three high-grade malignant tumors (an osteosarcoma, a metastasis of osteosarcoma, and a chondrosarcoma) were aneuploid by IC, but diploid by FCM. In the osteosarcomas this may be due to preoperative chemotherapy. The fact that aneuploidy was not detected by FCM may be due to chromosome losses counteracting the increased copy numbers of the chromosomes studied and to the change in the DNA content being too slight. Four benign tumors were aneuploid by IC, two of these were also aneuploid by FCM. All four represent tumor types which are potentially aggressive or premalignant. Thus, the results suggest that especially IC might be able to pinpoint those benign tumors with possible aggressive features.

The samples aneuploid by FCM were all except one (an osteosarcoma, # 17) aneuploid by IC (in this tumor IC was done with only one probe). In previous studies, IC and FCM (also image cytometry) have been concordant in 6 0 4 8 % of the cases studied (3,8,9,28). In the present study, IC and FCM results were concordant in 74% of the tumor specimens with respect to the presence or absence of aneuploidy. Taking into account the differences of these methods, this level of concordance is acceptable.

The concordance of the FCM results of successive samples from the same tumors was usually good (Table 2). Only in a case of fibrous dysplasia (# 4 ) were the results discordant. The FCM of a fresh sample of the first specimen was slightly suggestive of hyperdiploidy, which

could not be verified in a paraffin-embedded sample. The karyotype was normal, and IC did not reveal any NCA. The diploidy of the second sample could not, however, be verified from a repeated FCM on paraffin-embedded tissue, and the hyperdiploid DI of the second sample should be regarded as false-positive. Successive IC findings were concordant in one tumor (# 12) and discordant in the other (# 13). The reason why NCA was found in sample 13a but not in 13b remains somewhat unclear. However, the possibility of the second sample not being representative cannot be excluded. Clonal evolution seems a less likely explanation, as the samples were taken only 5 weeks apart.

The clonal aberrations detected with cytogenetic analysis usually agreed with IC and FCM. The cytogenetic analysis of a giant cell tumor (# 10) revealed loss of chromosome 7, which was not detected in IC. This may be explained by the possibility that the markers contained the centromeric region of chromosome 7. DI 1 .OO indicates that the loss of chromosomes from the C group was compensated with marker chromosomes of approximately the same size and that the net changes in DNA content were too small to be detected by FCM. In tumor # 21, the hypotriploid clone in cytogenetic analysis was not detected by FCM, which revealed only a hexaploid clone. This could be due to a selection in the cell culture or tumor heterogeneity. The opposite was seen in tumor # 22, which had hypodiploid and hypotetraploid clones by cytogenetics, but only a hypodiploid clone by FCM. In tumor # 21 and 23 the polyploidization of the hypotriploid clones detected in cytogeneitc analysis would result in lower CIS than the DIs. Similar findings of higher than expected DIs have been presented previously (24,31,39). Possible reasons for this are complex chromosomal aberrations affecting the total DNA content, marker chromosomes with high DNA copy number, heterogeneity of malignant tumors, and underestimation of the prevalence of double minutes in cytogenetics (40 ) .

Several malignant tumors had normal karyotypes. However, all these tumors were aneuploid by IC and/or FCM. Thus, it seems obvious that the normal karyotypes were artifacts from the culture and the metaphases did not represent tumor cells but normal stromal elements. This is a recognized problem in cytogenetic analysis.

Finally, it should be stated that the methods described here have some limitations. False-negative results can be obtained in cytogenetic analysis due to stromal over- growth. False-negative results can be obtained in IC if the aberrations are not within the scope of the probes used. In FCM, false-negative results can be obtained if thin paraffin-embedded samples are analyzed due to selective slicing of large, aneuploid nuclei (35 ) . It is commonly believed that fresh samples do not give false-negative results in FCM. As to false-positive results, it is the general opinion that undisputed clonal aberrations do not arise in the cell culture. With the 5% cutoff level used in this study, there should be no false-positive IC results based on our experience from control samples and on previous studies (4,7,8,12,28,30). False-positive results can be


seen in FCM if the sample is allowed to undergo autolysis (1 ). In the present study, however, the samples were analyzed or deep-frozen within an hour.

In conclusion, IC was the most sensitive method for detection of aneuploidy, even though it was usually performed with only two (peri)centromeric probes. The three methods provided complementary information of the tumors. When used simultaneously, these methods can improve the sensitivity for the detection of genomic changes in a tumor.

ACKNOWLEDGMENTS The technical assistance of Ms. Monica Schoultz is

gratefully acknowledged.

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Comparison of cytogenetics, interphase cytogenetics, and DNA flow cytometry in bone tumors