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Br J Cancer. 1994 August; 70(2): 255–262.
PMCID: PMC2033519

Molecular genetic analysis of flow-sorted ovarian tumour cells: improved detection of loss of heterozygosity.

Abstract

Detection of loss of heterozygosity (LOH) is usually performed on homogenised tumour specimens. In this type of analysis samples with a low percentage of tumour cells have to be excluded and possible intra-tumour heterogeneity is obscured. In this study we report the application of polymerase chain reaction (PCR)-driven LOH detection with in total 22 microsatellite markers for chromosome 1q, 3p, 3q, 4p, 6p, 6q, 11p, 11q, 17p, 17q, 18p, 18q, Xp and Xq on flow-sorted cells from fresh and paraffin-embedded ovarian tumour tissue. Titration experiments showed that LOH can be detected with as few as 100 cell equivalents of DNA. Clear examples of LOH could be detected in the sorted aneuploid fractions from one unilateral and two bilateral ovarian tumours from three patients. In two samples the sorted fraction was less than 10% of the total sample. The bilateral tumours from the same patient showed loss of identical alleles for one marker (case OV64) and two markers (case OV69), indicative of their monoclonal origin. Multiparameter flow cytometry using two different ovarian tumour markers (MOv18 and BMA180), an anti-cytokeratin monoclonal antibody (MAb) (M9), an anti-vimentin MAb (V9) and a MAb against the panepithelial antigen 17-1A on the fresh ascites cells of the fourth ovarian cancer patient was used to investigate possible intra-tumour heterogeneity. We showed the presence of at least three phenotypically different populations, of which the diploid, keratin-positive, vimentin-negative population showed a similar LOH pattern as the aneuploid population (DNA index = 1.7), indicative of its neoplastic origin. The same LOH pattern was shown in an omentum metastasis from this patient also having the same aneuploid DNA index of 1.7. The sharing of the same LOH pattern by the diploid and aneuploid tumour cell populations suggests that the observed allele loss events occurred before the development of aneuploidy. PCR on flow-sorted cells is thus an important tool to study clonal diversity in tumours.

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Selected References

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  • Bianchi AB, Navone NM, Conti CJ. Detection of loss of heterozygosity in formalin-fixed paraffin-embedded tumor specimens by the polymerase chain reaction. Am J Pathol. 1991 Feb;138(2):279–284. [PubMed]
  • Boynton RF, Blount PL, Yin J, Brown VL, Huang Y, Tong Y, McDaniel T, Newkirk C, Resau JH, Raskind WH, et al. Loss of heterozygosity involving the APC and MCC genetic loci occurs in the majority of human esophageal cancers. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3385–3388. [PubMed]
  • Burmer GC, Rabinovitch PS, Loeb LA. Frequency and spectrum of c-Ki-ras mutations in human sporadic colon carcinoma, carcinomas arising in ulcerative colitis, and pancreatic adenocarcinoma. Environ Health Perspect. 1991 Jun;93:27–31. [PMC free article] [PubMed]
  • Chenevix-Trench G, Leary J, Kerr J, Michel J, Kefford R, Hurst T, Parsons PG, Friedlander M, Khoo SK. Frequent loss of heterozygosity on chromosome 18 in ovarian adenocarcinoma which does not always include the DCC locus. Oncogene. 1992 Jun;7(6):1059–1065. [PubMed]
  • Coney LR, Tomassetti A, Carayannopoulos L, Frasca V, Kamen BA, Colnaghi MI, Zurawski VR., Jr Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein. Cancer Res. 1991 Nov 15;51(22):6125–6132. [PubMed]
  • Corver WE, Cornelisse CJ, Fleuren GJ. Simultaneous measurement of two cellular antigens and DNA using fluorescein-isothiocyanate, R-phycoerythrin, and propidium iodide on a standard FACScan. Cytometry. 1994 Feb 1;15(2):117–128. [PubMed]
  • Devilee P, van den Broek M, Kuipers-Dijkshoorn N, Kolluri R, Khan PM, Pearson PL, Cornelisse CJ. At least four different chromosomal regions are involved in loss of heterozygosity in human breast carcinoma. Genomics. 1989 Oct;5(3):554–560. [PubMed]
  • Foulkes WD, Campbell IG, Stamp GW, Trowsdale J. Loss of heterozygosity and amplification on chromosome 11q in human ovarian cancer. Br J Cancer. 1993 Feb;67(2):268–273. [PMC free article] [PubMed]
  • Foulkes WD, Ragoussis J, Stamp GW, Allan GJ, Trowsdale J. Frequent loss of heterozygosity on chromosome 6 in human ovarian carcinoma. Br J Cancer. 1993 Mar;67(3):551–559. [PMC free article] [PubMed]
  • Futreal PA, Söderkvist P, Marks JR, Iglehart JD, Cochran C, Barrett JC, Wiseman RW. Detection of frequent allelic loss on proximal chromosome 17q in sporadic breast carcinoma using microsatellite length polymorphisms. Cancer Res. 1992 May 1;52(9):2624–2627. [PubMed]
  • Hamilton TC, Young RC, McKoy WM, Grotzinger KR, Green JA, Chu EW, Whang-Peng J, Rogan AM, Green WR, Ozols RF. Characterization of a human ovarian carcinoma cell line (NIH:OVCAR-3) with androgen and estrogen receptors. Cancer Res. 1983 Nov;43(11):5379–5389. [PubMed]
  • Hedley DW, Friedlander ML, Taylor IW, Rugg CA, Musgrove EA. Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry. J Histochem Cytochem. 1983 Nov;31(11):1333–1335. [PubMed]
  • Hiddemann W, Schumann J, Andreef M, Barlogie B, Herman CJ, Leif RC, Mayall BH, Murphy RF, Sandberg AA. Convention on nomenclature for DNA cytometry. Committee on Nomenclature, Society for Analytical Cytology. Cancer Genet Cytogenet. 1984 Oct;13(2):181–183. [PubMed]
  • Hoffman RA, Kung PC, Hansen WP, Goldstein G. Simple and rapid measurement of human T lymphocytes and their subclasses in peripheral blood. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4914–4917. [PubMed]
  • Jones MH, Nakamura Y. Deletion mapping of chromosome 3p in female genital tract malignancies using microsatellite polymorphisms. Oncogene. 1992 Aug;7(8):1631–1634. [PubMed]
  • Lasko D, Cavenee W, Nordenskjöld M. Loss of constitutional heterozygosity in human cancer. Annu Rev Genet. 1991;25:281–314. [PubMed]
  • Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988 Feb 11;16(3):1215–1215. [PMC free article] [PubMed]
  • Miotti S, Canevari S, Ménard S, Mezzanzanica D, Porro G, Pupa SM, Regazzoni M, Tagliabue E, Colnaghi MI. Characterization of human ovarian carcinoma-associated antigens defined by novel monoclonal antibodies with tumor-restricted specificity. Int J Cancer. 1987 Mar 15;39(3):297–303. [PubMed]
  • Mok CH, Tsao SW, Knapp RC, Fishbaugh PM, Lau CC. Unifocal origin of advanced human epithelial ovarian cancers. Cancer Res. 1992 Sep 15;52(18):5119–5122. [PubMed]
  • Pak KY, Nedelman MA, Fogler WE, Tam SH, Wilson E, Van Haarlem LJ, Colognola R, Warnaar SO, Daddona PE. Evaluation of the 323/A3 monoclonal antibody and the use of technetium-99m-labeled 323/A3 Fab' for the detection of pan adenocarcinoma. Int J Rad Appl Instrum B. 1991;18(5):483–497. [PubMed]
  • Phillips N, Ziegler M, Saha B, Xynos F. Allelic loss on chromosome 17 in human ovarian cancer. Int J Cancer. 1993 Apr 22;54(1):85–91. [PubMed]
  • Radford DM, Fair K, Thompson AM, Ritter JH, Holt M, Steinbrueck T, Wallace M, Wells SA, Jr, Donis-Keller HR. Allelic loss on a chromosome 17 in ductal carcinoma in situ of the breast. Cancer Res. 1993 Jul 1;53(13):2947–2949. [PubMed]
  • Schaafsma HE, Ramaekers FC, van Muijen GN, Lane EB, Leigh IM, Robben H, Huijsmans A, Ooms EC, Ruiter DJ. Distribution of cytokeratin polypeptides in human transitional cell carcinomas, with special emphasis on changing expression patterns during tumor progression. Am J Pathol. 1990 Feb;136(2):329–343. [PubMed]
  • Schueler JA, Cornelisse CJ, Hermans J, Trimbos JB, van der Burg ME, Fleuren GJ. Prognostic factors in well-differentiated early-stage epithelial ovarian cancer. Cancer. 1993 Feb 1;71(3):787–795. [PubMed]
  • Sharpless T, Traganos F, Darzynkiewicz Z, Melamed MR. Flow cytofluorimetry: discrimination between single cells and cell aggregates by direct size measurements. Acta Cytol. 1975 Nov-Dec;19(6):577–581. [PubMed]
  • Smit VT, Fleuren GJ, van Houwelingen JC, Zegveld ST, Kuipers-Dijkshoorn NJ, Cornelisse CJ. Flow cytometric DNA-ploidy analysis of synchronously occurring multiple malignant tumors of the female genital tract. Cancer. 1990 Oct 15;66(8):1843–1849. [PubMed]
  • Stein R, Goldenberg DM, Mattes MJ. Normal tissue reactivity of four anti-tumor monoclonal antibodies of clinical interest. Int J Cancer. 1991 Jan 21;47(2):163–169. [PubMed]
  • Sundaresan V, Ganly P, Hasleton P, Bleehen NM, Rabbitts P. Paraffin wax-embedded material as a source of DNA for the detection of somatic genetic changes. J Pathol. 1993 Jan;169(1):43–52. [PubMed]
  • van den Ingh HF, Griffioen G, Cornelisse CJ. Flow cytometric detection of aneuploidy in colorectal adenomas. Cancer Res. 1985 Jul;45(7):3392–3397. [PubMed]
  • Van Muijen GN, Ruiter DJ, Warnaar SO. Coexpression of intermediate filament polypeptides in human fetal and adult tissues. Lab Invest. 1987 Oct;57(4):359–369. [PubMed]
  • van Niekerk CC, Boerman OC, Ramaekers FC, Poels LG. Marker profile of different phases in the transition of normal human ovarian epithelium to ovarian carcinomas. Am J Pathol. 1991 Feb;138(2):455–463. [PubMed]
  • Vindeløv LL, Christensen IJ, Nissen NI. Standardization of high-resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards. Cytometry. 1983 Mar;3(5):328–331. [PubMed]
  • Weber JL, May PE. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet. 1989 Mar;44(3):388–396. [PubMed]

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