Breast Cancer Research and Treatment

, Volume 23, Issue 3, pp 201–210 | Cite as

Chromosomein situ hybridization on formalin-fixed mammary tissue using non-isotopic, non-fluorescent probes: technical considerations and biological implications

  • Kapil Dhingra
  • Aysegul Sahin
  • Janet Supak
  • S. Y. Kim
  • Gabriel Hortobagyi
  • Walter N. Hittelman


Fluorescentin situ hybridization techniques have provided an important tool for interphase cytogenetic studies of human neoplasms. However, these techniques are difficult to use on formalin-fixed archival tissue sections. We describe here a non-fluorescent, non-isotopicin situ hybridization (ISH) approach that is easily applicable to paraffin-embedded breast tissue sections. The technical steps that must be monitored and individualized to optimize signal generation and detection are discussed. This ISH technique has several advantages over fluorescent detection methods. The signal obtained can be viewed using an ordinary light microscope and does not fade with time. More importantly, the signal is observed and analyzed in the context of tissue morphology. The technique permits detection of numerical chromosomal abnormalities not only in malignant but also in apparently normal and potentially premalignant mammary tissue. This may allow identification of focal genetic abnormalities as well as field-defects and enable analysis of their evolution during the multistep transformation to mammary neoplasm. This technique is also suitable for analysis of tumor heterogeneity and the correlation of numerical chromosomal aberrations with histologic, immunocytochemical, and clinical features of breast tumors.

Key words

breast cancer in situ hybridization interphase cytogenetics paraffin-embedded sections multistep carcinogenesis tumor cytogenetics 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Nowell PC, Hungerford DA: A minute chromosome in human chronic granulocytic leukemia. Science 132: 1497–1499, 1960Google Scholar
  2. 2.
    Rowley JD: Molecular cytogenetics: Rosetta stone for understanding cancer (Twenty ninth G.H.A. Clowes Memorial Award Lecture). Cancer Res 50: 3816–3825, 1990PubMedGoogle Scholar
  3. 3.
    Gebhart E, Bruderlein S, Augustus M, Siebert E, Feldner E, Schmidt W: Cytogenetic studies on human breast carcinomas. Breast Cancer Res Treat 8: 125–138, 1986PubMedGoogle Scholar
  4. 4.
    Mitelman F: Clustering of chromosomal breakpoints in neoplasia. Cancer Genet Cytogenet 19: 67–71, 1986PubMedGoogle Scholar
  5. 5.
    Pinkel D, Landegent J, Collins C, Fuscoe J, Segraves R, Lucas J, Gray J: Fluorescencein situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci USA 85: 9138–9142, 1988PubMedGoogle Scholar
  6. 6.
    Pinkel D, Straume T, Gray JW: Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83: 2934–2938, 1986PubMedGoogle Scholar
  7. 7.
    Devilee P, Thierry RF, Kievits T, Kolluri R, Hopman AHN, Willard HF, Pearson PL, Cornelisse CJ: Detection of chromosome aneuploidy in interphase nuclei from human primary breast tumors using chromosome-specific repetitive DNA probes. Cancer Res 48: 5825–5830, 1988PubMedGoogle Scholar
  8. 8.
    Hopman AHN, Poddighe PJ, Smeets AWGB, Moesker O, Beck JLM, Vooijs GP, Ramaekers FCS: Detection of numerical chromosome aberrations in bladder cancer byin situ hybridization. Am J Pathol 135: 1105–1117, 1989PubMedGoogle Scholar
  9. 9.
    Hopman AHN, Moesker O, Smeets AWGB, Pauwels RPE, Vooijs GP, Ramaekers FCS: Numerical chromosome 1, 7, 9 and 11 aberrations in bladder cancer detected byin situ hybridization. Cancer Res 51: 644–651, 1991PubMedGoogle Scholar
  10. 10.
    Poddighe PJ, Moesker O, Smeets D, Awwad BH, Ramaekers FCS, Hopman AHN: Interphase cytogenetics of hematological cancer: Comparison of classical karyotyping andin situ hybridization using a panel of eleven chromosome specific DNA probes. Cancer Res 51: 1959–1967, 1991PubMedGoogle Scholar
  11. 11.
    Warrell RP, Frankel SR, Miller WH, Scheinberg DA, Itri LM, Hittelman WN, Vyas R, Andreeff M, Tafuri A, Jakubowski A, Gabrilove J, Gordon MS, Dmitrovsky E: Differentiation therapy of acute promyelocytic leukemia with Tretinoin (all-trans retinoic acid). N Engl J Med 324: 1385–1393, 1991PubMedGoogle Scholar
  12. 12.
    Tkachuk DC, Westbrook CA, Andreeff M, Donlon TA, Cleary ML, Suryanarayan K, Homge M, Redner A, Gray J, Pinkel D: Detection ofbcr-abl fusion in chronic myelogenous leukemia byin situ hybridization. Science 250: 559–562, 1990PubMedGoogle Scholar
  13. 13.
    Waldman FM, Carroll PK, Kerschmann R, Cohen MB, Field FG, Mayall BH: Centromeric copy number of chromosome 7 is strongly correlated with tumor grade and labeling index in human bladder cancer. Cancer Res 51: 3807–3813, 1991PubMedGoogle Scholar
  14. 14.
    Arnoldus EPJ, Dreef EJ, Noordermeer IA, Verheggen MM, Thierry RF, Peters ACB, Cornelisse CJ, Van der Ploeg M, Raap AK: Feasibility ofin situ hybridization with chromosome specific DNA probes on paraffin wax embedded tissue. J Clin Pathol 44: 900–904, 1991PubMedGoogle Scholar
  15. 15.
    Van der Loos CM, Volkers HH, Rook R, Van den Berg FM, Houthoff HJ: Simultaneous application ofin situ DNA hybridization and immunohistochemistry on one tissue section. Histochem J 21: 279–284, 1989PubMedGoogle Scholar
  16. 16.
    Burns J, Graham AK, McGee JO: Non-isotopic detection ofin situ nucleic acid in cervix: an updated protocol. J Clin Pathol 41: 897–899, 1988PubMedGoogle Scholar
  17. 17.
    Mullink H, Walboomers MM, Tadema TM, Jansen DJ, Meijer CJLM: Combined immuno- and non-radioactive hybridochemistry on cells and tissue sections: Influence of fixation, enzyme pretreatment, and choice of chromogen on detection of antigen and DNA sequences. J Histochem Cytochem 37: 603–609, 1989PubMedGoogle Scholar
  18. 18.
    Emmerich P, Jauch A, Hofman MC, Cremer T, Walt H: Interphase cytogenetics in paraffin embedded sections from human testicular germ cell tumor xenografts and in corresponding cultured cells. Lab Invest 61: 235–242, 1989PubMedGoogle Scholar
  19. 19.
    Hopman AHN, van Hooren E, van de Kaa CA, Vooijs GP, Ramaekers FCS: Detection of numerical chromosome aberrations usingin situ hybridization in paraffin sections of routinely processed bladder cancers. Mod Pathol 4: 503–513, 1991PubMedGoogle Scholar
  20. 20.
    Hopman AHN, Ramaekers FCS, Raap AK, Beck JLM, Devilee P, Van der Ploeg M, Vooijs GP:In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors. Histochemistry 89: 307–316, 1988PubMedGoogle Scholar
  21. 21.
    Fearon ER, Vogelstein B: A genetic model for colorectal tumorigenesis. Cell 61: 759–767, 1990PubMedGoogle Scholar
  22. 22.
    Bischoff FZ, Yim SO, Pathak S, Grant G, Siciliano MJ, Giovanella BC, Strong LC, Tainsky MA: Spontaneous abnormalities in normal fibroblasts from patients with Li-Fraumeni cancer syndrome: Aneuploidy and immortalization. Cancer Res 50: 7979–7984, 1990PubMedGoogle Scholar
  23. 23.
    Dupont WD, Page DL: Risk factors for breast cancer in women with proliferative breast disease. N Engl J Med 312: 146–151, 1985PubMedGoogle Scholar
  24. 24.
    Page DL, Dupont WD, Rogers LW, Landenberger M: Intraductal carcinoma of the breast: Follow-up after biopsy only. Cancer 49: 751–758, 1982PubMedGoogle Scholar
  25. 25.
    Rosen PP, Braun DW Jr, Kinne DW: The clinical significance of preinvasive breast carcinoma. Cancer 46: 919–925, 1980PubMedGoogle Scholar
  26. 26.
    Kiang DT: Chemoprevention of breast cancer: Are we ready? J Natl Cancer Inst 83: 462–463, 1991PubMedGoogle Scholar
  27. 27.
    Rodgers CS, Hill SM, Hulten MA: Cytogenetic analysis in human breast carcinoma. I. Nine cases in the diploid range investigated using direct preparations. Cancer Genet Cytogenet 13: 95–119, 1984PubMedGoogle Scholar
  28. 28.
    Mars W, Saunders GF: Chromosomal abnormalities in breast cancer. Cancer Metastasis Rev 9: 35–43, 1990PubMedGoogle Scholar
  29. 29.
    Dutrillaux B, Gerbault-Seureau M, Zafrani B: Characterization of chromosomal anomalies in human breast cancer — A comparison of 30 paradiploid cases with few chromosome changes. Cancer Genet Cytogenet 49: 203–217, 1990PubMedGoogle Scholar
  30. 30.
    Hall JM, Lee MK, Newman B, Morrow JE, Anderson LA, Huey B, King M-C: Linkage of early-onset familial breast cancer to chromosome 17q21. Science 250: 1684–1689, 1990PubMedGoogle Scholar
  31. 31.
    Cherif D, Bernard O, Berger R: Detection of single-copy genes by nonisotopicin situ hybridization on human chromosomes. Hum Genet 81: 358–362, 1989PubMedGoogle Scholar
  32. 32.
    Pequignot EV, Dutrillaux B, Magdelenat H, Moisan MC: Mapping of single-copy DNA sequences on human chromosomes byin situ hybridization with biotinylated probes: Enhancement of detection sensitivity by intensified-fluorescence digital-imaging microscopy. Proc Natl Acad Sci USA 86: 582–586, 1989PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1992

Authors and Affiliations

  • Kapil Dhingra
    • 1
  • Aysegul Sahin
    • 2
  • Janet Supak
    • 1
  • S. Y. Kim
    • 1
  • Gabriel Hortobagyi
    • 1
  • Walter N. Hittelman
    • 1
  1. 1.Section of Breast Medical Oncology/Box 56The University of Texas M.D. Anderson Cancer CenterHoustonUSA
  2. 2.Department of PathologyThe University of Texas M.D. Anderson Cancer CenterHoustonUSA

Personalised recommendations