Detection of Somatic DNA Alterations in Ovarian Cancer by DNA Fingerprint Analysis Philip Chen, PhD,* Terence Hurst, BSc,t and Soo-Keat Khoo, MDt

The M13 phage single-strand DNA probe which recognizes highly polymorphic loci was applied to HinfI-digested DNA isolated from tumor tissue and peripheral leukocytes from 20 patients with ovarian cancer. An average of 22 minisatellite- containing DNA fragments were observed per individual. DNA fingerprint analysis revealed a change in restriction-fragment-length patterns in the DNA from 12 of 20 (60%) tumors compared with the patient’s constitutional DNA. Deletion of one or more bands from the tumor was recognized by the probe in seven cases, new bands were identified in two, and intensity shift was demonstrated in eight. The authors conclude that the unmapped M13 minisatellite probe is a useful method for identifying cancer-related somatic DNA alterations. Cancer 67:1551-1555,1991.

T IS GENERALLY ACCEPTED that tumors arise I through the accumulation of several genetic changes affecting cell proliferation;’ the degree of change is asso- ciated with the aggressive nature of the tumor.2 Chro- mosomal and genetic instability is known to be important and may be characteristic of tumor deve l~pment .~?~ So- matic variations in tumors resulting from major chro- mosomal rearrangements can be demonstrated by karyo- type analysis,’ but it is technically difficult to obtain chro- mosomal preparations from solid tumors. DNA analysis can now be used to detect somatic variations, in particular to demonstrate chromosomal loss or somatic heterozy- gosity. With the use of restriction-fragment-length poly- morphism analysis, an allelic loss in a particular chro- mosome region has been shown in tumors such as reti- noblastoma, Wilms’ tumor, and ’ Most of these marker systems are, however, dimorphic, and the detection of somatic changes in tumor cells is limited by their relatively low variability. This problem is overcome by the recent development of DNA fingerprint analysis; the fingerprints obtained by this technique represent mul- tiple hypervariable fragments derived from autosomal loci dispersed throughout the genome. 12-14

From the *Molecular Oncology, Queensland Institute of Medical Re- search, and the ?Department of Obstetrics and Gynecology, University of Queensland, Bnsbane, Australia.

Supported by grants from the Queensland Cancer Fund. The authors thank Dr. M. Lavin for helpful discussions, B. Sanderson

for technical assistance, and H. Matthews for photography. Address for reprints: Philip Chen, PhD, Queensland Institute of Med-

ical Research, Bramston Terrace, Herston, Brisbane, Australia 4006. Accepted for publication September 1, 1990.

With this technique, somatic changes have been re- ported in various human cancers, 1 5 - 1 7 including one ovarian tumor.’* We analyzed the constitutional and tu- mor DNA of 20 patients with ovarian cancer and observed somatic changes in the tumor DNA in 12 of them. The changes included alterations in the relative intensities of hybridizing DNA fragments and loss or gain of bands.

Materials and Methods

Patients

The study tested 20 patients (some including family members) with epithelial cancer of the ovary, aged 18 to 83 years. The diagnosis in each case was confirmed by histologic examination of paraffin-embedded sections by Dr. G. Wright, histopathologist at the Royal Women’s Hospital. Samples of tumor tissue and peripheral venous blood were obtained before the commencement of che- motherapy or radiation therapy. The fresh tumor tissue was dissected free of fat and connective tissue, mechani- cally dispersed, and separated from blood cells by Ficoll- paque gradient and lysis (Pharmacia Inc., Piscataway, NJ). The peripheral leukocytes also were separated by Ficoll- paque gradient. DNA was isolated from the tumor tissue and blood by the method previously described by Muller et al. l 9

DNA AnaIysis Constitutional and tumor DNA (2 to 4 pg) from each

individual was digested overnight at 37°C with 20 units of HinfI under conditions recommended by the manu- facturers (New England Biolabs, Beverly, MA). The di-

1551

1552 CANCER March 15 199 1 Vol. 67

gested DNA of each patient underwent electrophoresis in adjacent lanes through a 25-cm long 0.6% agarose gel (Sea Kem GTG; FMC BioProducts, Rockland, ME) at 40 V for approximately 66 hours at room temperature.*’ The DNA fragments were transferred to a nylon membrane (Amersham Hybond-N) by southern blotting overnight using a 20 times concentrated solution of SSC (single- concentrated SSC is 0.15 mol/l of NaCl and 15 mmol/l of trisodium citrate). The membranes were baked at 80°C in a vacuum oven for 2 hours.

Prehybridization was done as previously described.’’ Hybridization was done in the same solution at 55°C for 24 hours with the addition of a ”P-deoxycytidine tri- phosphate-labeled probe; 250 ng M 13mp8 single-stranded DNA (New England Biolabs) was labeled by the method of Feinberg and Vogelstein22 at room temperature for 2 hours. Unincorporated radionucleotides were removed by passing the probe through a column (NACS PREPAC, Bethesda Research Laboratories, Gaithersburg, MD). Af- ter hybridization, the filters were washed for 30 minutes in twice-concentrated SSC with 0.1 % sodium dodecyl sul- fate at 55°C and autoradiographed with two intensifying screens for 1 to 2 days.

The patterns of bands in immediately adjacent lanes were compared visually on a light box. They were scored directly from original autoradiographs taken at short, me- dium, and long exposures. In most cases, the experiments were repeated at least twice.

Results

Comparison of Constitutional and Tumor DNA

In this study, we analyzed the DNA from 20 patients with ovarian cancer, most in an advanced stage of the disease. Overall, alterations in DNA fingerprints were ob- served in 12 of these patients (Table 1). Examples of tu- mors showing somatic changes are illustrated in Figure 1. There were changes in the relative intensities of mini- satellite DNA fragments and loss and/or appearance of bands in tumor DNA compared with the corresponding constitutional DNA (Table 2). These changes were inter- preted as extensive rearrangements of chromosomal ma- terial in the tumor cells, as previously reported.’5316 The average number of somatic changes was calculated to be 1.70 (34 in 20 patients) fragments per patient.

In four patients there were changes in relative intensities and loss of fragments (Fig. 1). One patient (19/88) lost four bands in the tumor DNA fingerprint, of approximate sizes 22, 21, 20, and 18 kilobases (kb), had a reduction in hybridization intensity in two bands of sizes 15 and 12 kb, and had an increased signal of a band at 9.2 kb. One patient (46/88) lost one band at 27 kb and had an intensity shift at 26 and 9.5 kb. Another patient (35/87) lost one

TABLE 1 . DNA Fingerprint Analysis in 20 Cases of Human Ovarian Cancer

Tumor DNA fingerprint

Age FIGO DNA Bands New Intensity Patient (yr) stage source lost bands shift

-* - 25/87 77 I11 T-1B - - AF

T-IB 1 T-ID 1 T-1E 1 AF

- -

35/87 58 IIIC T-IA I t -

- ~ - - - 12/88 52 I11 T-1B

- 19/88 63 IV PD 4 31/88 51 I11 ov

45/88 59 111 ov 5 1 A F

46/88 66 111 AF 1 12/89 44 111 ov 14/89 72 111 T-1A -

17/89 68 I11 T-IB - 19/89 67 I1 ov 22/89 18 IV T - -

17/88 68 111 T-1B 1 1 20/89 83 I11 T-1E 36/89 63 111 T-ID 3 52/89 57 111 T-IB 1

- -

- 40/88 58 111 T-ID ~

- - 41/88 61 111 T-IB

- -

-

- -

- - 13/89 62 IV PL-F ~

~

- -

- - 21/89 56 Ia A F

- -

-

-

FIGO: International Federation of Gynecology and Obstetrics; T-1A: right ovary; T-1B: left ovary; T-ID: omenturn; T-1E: left pelvic mass; A F ascites fluid; PD: peritoneal deposits; PL-F pleural fluid; OV: ovary of unknown side; T: abdominal tissue.

* Absence. t Numerals show number of band changes in the tumor DNA fin-

gerprints.

band at 8.2 kb and had an increased signal in the bands at 7.5 and 7.2 kb. The tumor DNA ofpatient 45/88 gained a band at 13.5 kb and lost bands of approximately 28, 25,21.5, 14.8, and 9.8 kb; no somatic changes were found in the malignant cells from the ascitic fluid (Fig. I) .

In one patient with metastatic disease, the tumor DNA was analyzed in samples from different sites. As shown in Figure 2, similar changes from the constitutional DNA were observed in all tumor DNA isolated except that from malignant cells from ascitic fluid. There was a reduction in hybridization intensity in two bands at 7.5 and 7.2 kb, and a loss of one band at 8.2 kb.

In two patients, their daughters were available for study. In patient 25/87 a fragment (1 2.2 kb) that was inherited by her daughter showed somatic change in the tumor DNA. Two daughters of patient 19/89 were included in the DNA fingerprint analysis. Two bands (27 and 20.8 kb), appearing as tumor DNA alterations in the mother’s sample, were detected in an unchanged form in the second daughter DNA (D2, Fig. 3).

No. 6 DNA ALTERATIONS IN OVARIAN CANCER - Chen et al. 1553

FIG. 1. DNA fingerprints showing loss and appearance of bands and changes in relative intensities in malignant ovarian tu- mors. DNA samples from peripheral blood leukocytes (lanes C), tumor tissues (lanes T), and ascitic fluid (lane F) were obtained from Patients 19/88,46/88,45/88, and 14/ 89. The top bands (28 and 25 kb) of 45/88 were evaluated on a less heavily exposed autoradiograph. The faint bands appear at lanes T of Patients 19/88 and 46/88 are due to contamination oftumor cells with normal stromal cells. Changes in the minisatellite pattern are great and DNA fragment length in kilobase pairs is indicated on the right of the autoradiograms.

Clinical Correlation

The series was too small for detailed correlation. How- ever, somatic changes were only observed in the tumor DNA from patients aged 50 years and over (Table 1). Neither patient younger than 50 years of age showed changes. Somatic changes in the tumor DNA were found in four of seven patients aged 50 to 59 years, six of eight patients aged 60 to 69 years, and two of three patients aged 70 years and older.

The clinical significance of these changes is unclear be- cause the follow-up was less than 12 months. However, there was one patient (19/88) who showed a rapid pro- gression of disease within 6 months; her tumor DNA had marked changes with loss of four bands and an intensity shift of three bands (Fig. 1).

may not be accessible to all groups, and the method has technical difficulties. The primary advantage of the M 13 probe is its commercial availability.

There is evidence that cancer cells show substantial ge- netic alterations. Previous studies show that DNA finger- prints of peripheral leukocytes, normal mucosa cells, and spermatozoa from the same individual are ~ imi l a r . ‘~” ’ ’~~ Therefore, variations in tumor DNA fingerprints different from those of corresponding constitutional DNA of the patients with cancer are unlikely to be tissue specific; these changes represent somatic mutations. By comparing DNA

TABLE 2. Changes in Tumor DNA Fingerprints in Ovarian Cancer*

Genetic No. of patients No. of fragment change (n = 20) changes

Discussion

The technique of DNA fingerprint analysis, developed by Jeffreys et a1.,’* has become widely used in genetic examination of many species. This advanced technology

Intensity 8 16 Lost bands I 16 New bands 2 2 No change 8 0

* The data are derived from Table 1.

1554 CANCER March 15 199 1 Vol. 67

more were shown in eight cases. These results indicate that ovarian tumor cells possess genomic instability, con- firming earlier findings by Smit et al." in one ovarian tumor, and those in other tumors such as colorectal can- cer, l 5 bladder cancer,23 Wilms' tumour, malignant mel- anoma, Burkitts' lymphoma, and Merkel cell carcinoma. l7

FIG. 2. DNA fingerprint analysis of tumor samples from different sites in Patient 35/87. Lane A: blood leukocytes; B: right ovary; C left ovary; D: greater omentum; E: left pelvic mass; F cells from ascitic fluid. With the exception of lane F, there was a consistent decrease in intensity in bands of 7.5 and 7.2 kb and a loss of band 8.2 kb in the other tumor sites compared with the constitutional DNA.

fingerprints from tumor-constitutional Pairs in ovarian cancer, we found somatic changes in the tumor DNA in

FIG. 3. DNA fingerprint analysis of Patient 19/89 and her offspring. C: constitutional DNA from blood leukocytes; T: tumor; D,, Dz: blood

12 of 20 patients. Deletion of one or bands from the tumors was demonstrated in Seven cases; in two a new band was identified. Intensity shifts of one band or

leukocytes of daughter I and daughter 2, respectively. A change of hy- bridization intensity was seen in bands 27 and 20.8 in the tumor DNA. AH of these bands also were present in the DNA of her second daughter (D2).

No. 6 DNA ALTERATIONS IN OVARIAN CANCER * Chen et a/. 1555

Loss of heterozygosity could be important in tumor de- velopment, as suggested in the case of tumor suppressor genes.24 DNA fingerprint analysis appears to detect genetic instability and chromosomal changes that may not be de- tected by the cytogenetic method.

To date, most human tumors investigated have been shown to be m o n o ~ l o n a l . ~ ~ - ~ ~ DNA fingerprinting pro- vides an approach for the detection of clonal somatic mu- tations in tumors. Fey el al.29 found clonal abnormalities in 29 of 44 gastrointestinal tumors using a panel of three DNA fingerprinting probes: 33.5, 33.6, and a-globin 3’HVR. However, one disadvantage of DNA fingerprint- ing probes is the need to clone the normal homologues of alleles deleted in the tumor DNA to permit their lo- calizations in the genome. The results of our previous studies on a range of tumors” and of the current study on ovarian cancer support the utility of the M 13 probe for determining clonality in tumors; it may also be helpful in genotypic analysis of tumor progression.

In conclusion, we suggest that unmapped M 13 mini- satellite hybridizing sequences are a useful method for differentiating ovarian cancer-related somatic DNA al- terations. This approach may be able to identify specific DNA fragments but require considerable work in isola- tion, molecular cloning, and chromosomal map assign- ment of these tumor-associated DNA fragrnent~.~’ The cloned minisatellite may detect genomic changes in tumor progression, and concomitant use of other minisatellite families could examine molecular changes associated with ovarian tumorigenesis. In addition, DNA fingerprinting is a very useful tool for assessing tumor clonality.

REFERENCES

I . Klein G, Klein E. Evolution of tumors and the impact of molecular

2. Nowell PC. Molecular events in tumor development. N Engl J

3. Cairns J. The origin ofhuman cancers. Nature 198 1; 289:353-357 4. Sandberg AA, Turc-Carel C, Genemell RM. Chromosomes in solid

tumors and beyond. Cancer Res 1988; 48:1049-1059. 5 . Yunis JJ. The chromosomal basis of human neoplasia. Science

1983; 22 1:227-236. 6 . Cavenee WK, Dryja TP, Phillips RA et a/. Expression of recessive

alleles by chromosomal mechanisms in retinoblastoma. Nature 1983; 305:779-784.

7. Fearon ER, Vogelstein B, Feinberg AP. Somatic deletion and du- plication of genes on chromosome 1 I in Wilms’ tumors. Nature 1984; 309:176-178.

8. Koufos A, Hansen MF, Lampkin BC et a1. Loss of alleles at loci on human chromosome 1 1 during genesis of Wilms’ tumor. Nature 1984; 309: 170- 1 72.

9. Orkin SH, Goldman DS, Sallan SE. Development of homozygosity

oncology. Nature 1985; 315:190-195.

Med 1988; 319575-576.

for chromosome 1 Ip markers in Wilms’ tumor. Narzrre 1984; 309: 172- 174.

10. Reeve AE, Housiaux PJ, Gardner RJM, Chewings WE, Grindley RM, Millow LJ. Loss of a Harvey rus allele in sporadic Wilms’ tumour. Nature 1984; 309:174-176.

11. Dracopoli NC, Houghton AM, Old LJ. Loss of polymorphic re- striction fragments in malignant melanoma: Implication for tumor het- erogeneity. Proc Nut1 Acad Sci U S A 1985; 82: 1470-1474.

12. Jeffreys AJ, Wilson V, Thein SL. Hypervariable minisatellite re- gions in human DNA. Narure 1985; 314:67-73.

13. Nakamura Y , Leppert M, OConnell R ef ul. Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 1987; 235: 16 16-1622.

14. Vassart G, Georges M, Monsieur R, Brocas H, Lequarre AS, Christophe D. A sequence in the wild type M I3 bacteriophage detects hypervariable minisatellites in human and animal DNA. Science 1987;

15. Thein SL, Jeffreys AJ, Gooi HC el a/. Detection of somatic changes in human cancer DNA by DNA fingerprint analysis. Br J Cancer 1987; 55:353-356.

16. De Jong D, Voetdijk BMH, Kluin-Nelemans JC, van Ommen GJB, Kluin PM. Somatic changes in B-lymphoproliferative disorders (B- LPD) detected by DNA-fingerprinting. Br J Cancer 1988; 58:773-773.

17. Hayward N, Chen P, Nancarrow D et al. Detection of somatic mutations in tumors of diverse types by DNA fingerprinting with MI3 phage DNA. Int J Cancer 1990; 445:687-690.

18. Smit VTHB, Cornelisse CJ, De Jong P, Dijkshoorn NJ, Peters AAW, Fleuren GJ. Analysis of tumor heterogeneity in a patient with synchronously occurring female genital tract malignancies by DNA flow cytometry, DNA fingerprinting, and immunohistochemistry. Cancer

19. Muller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 121 5.

20. Chen P. Hayward NK, Kidson C, Ellem KAO. Conditions for generating well-resolved human DNA fingerprints using M 13 phage DNA. Niickeic Acids Res 1990; 18: 1065.

21. Church GM, Gilbert W. Genomic sequencing. Proc Nut1 Acad

22. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem

23. Uchida T, Neuwirth H, Cadavid NG. DNA fingerprints detect somatic DNA changes in transitional carcinoma of the bladder (TCC) (Abstr). Proceedings of the American Association of Cancer Research 1989; 30:314.

24. Klein G. The approaching era of the tumor suppressor gene. Sci- ence 1987; 238:1539-1544.

25. Fialkow PJ, Gartler SM, Yoshida A. Clonal origin of chronic myelocytic leukemia in man. Proc Nut/ Acad Sci U S A 1967; 58: 1468- I47 1.

26. Smith JW, Townsend DE, Sparkes RS. Genetic variants of glucose- 6-phosphate dehydrogenase in the study of carcinoma of the cervix. Cancer 1971; 28529-532.

27. Fearon ER, Hamilton SR, Vogelstein B. Clonal analysis of human colorectal tumors. Science 1987; 238: 193- 197.

28. Vogelstein B, Fearon ER, Hamilton SR, Feinberg AP. Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors. Science 1985; 227:642-645.

29. Fey MF, Well RA, Wainscoat JS, Thein SL. Assessment of clon- ality in gastrointestinal cancer by DNA fingerprinting. J Clin Invessl 1988;

30. Wong Z , Wilson V, Patel I, Povey S, Jeffreys AJ. Characterization ofa panel of high variable minisatellites cloned from human DNA. Ann H i m Gerzef 1987; 5 I :269-288.

235:683-684.

1988; 62:1146-1152.

9.i U S A 1984; 81:1991-1995.

1984; 1237~266-267.

82: 1532-1 537.