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Molecular Biologic Substaging of Stage I NSCLC Through Immunohistochemistry Performed on Formalin-Fixed, Paraffin-Embedded Tissue

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Lung Cancer

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 75))

Abstract

Non-small cell lung cancer (NSCLC) is the most common cause of death by malignancy in both men and women in the United States (1). The current staging system for NSCLC considers the size and location of the primary tumor (T), the involvement of regional lymph nodes (N), and the presence of distant metastases (M) (2). The standard treatment of patients with stage I NSCLC (T1-2N0M0) is resection of the primary tumor with no adjuvant therapy. However, even after complete resection, the 5-yr survival rate is only 55–72%, mainly due to the development of distant metastases (27). Therefore, a significant number of patients are under-staged and may benefit from adjuvant therapy.

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References

  1. Landis, S. H., Murray, T., Bolden, S., and Wingo, P. A. (1999) Cancer statistics, 1999. CA Cancer J. Clin. 49, 8–31.

    Article  PubMed  CAS  Google Scholar 

  2. Mountain, C. F. (2000) The international system for staging lung cancer. Semin. Surg. Oncol. 18(2), 106–115.

    Article  PubMed  CAS  Google Scholar 

  3. Harpole, D. H., Jr., Herndon, J. E., II, Young, W. G., Wolf, W. G., and Sabiston, D. C., Jr. (1995a) A prognostic model of recurrence and death in stage I non-small cell lung cancer utilizing presentation, histopathology and oncoprotein expression. Cancer Res. 55, 51–56.

    PubMed  CAS  Google Scholar 

  4. Mountain, C. F. (1997) Revisions in the international system for staging lung cancer. Chest 111, 1710–1717.

    Article  PubMed  CAS  Google Scholar 

  5. Mountain, C. F., Lukeman, J. M., and Hammar, S. P. (1987) Lung cancer classification: the relationship of disease extent and cell type to survival in a clinical trials population. J. Surg. Oncol. 35, 147–156.

    Article  PubMed  CAS  Google Scholar 

  6. Mountain, C. F. (1989) Value of the new TNM staging system for lung cancer. Chest 96, 47S–49S.

    PubMed  CAS  Google Scholar 

  7. Nesbitt, J. C., Putnam, J. B., Jr., Walsh, G. L., Roth, J. A., and Mountain, C. F. (1993) Survival in early-stage I non-small cell lung cancer? J. Thorac. Cardiovasc. Surg. 106, 90–94.

    Google Scholar 

  8. Ichinose, Y., Hara, N., Ohta, M., Yano, T., Maeda, K., Asoh, H., et al. (1993) Is T factor of the TNM staging system a predominant prognostic factor in pathologic stage I non-small cell lung cancer? J. Thorac. Cardiovasc. Surg. 106, 90–94.

    PubMed  CAS  Google Scholar 

  9. Macchiarini, P., Fontanini, G., Hardin, M. J., Chuanchieh, G., Bigini, D., Vignati, S., et al. (1993) Blood vessel invasion by tumor cells predicts recurrence in completely resected T1 N0M0 non-small cell lung cancer. J. Thorac. Cardiovasc. Surg. 106, 80–89.

    PubMed  CAS  Google Scholar 

  10. Thomas, P. A. and Rubinstein, L. (1990) Cancer recurrence after resection: T1N0 non-small cell lung cancer. Ann. Thorac. Surg. 49, 242–247.

    Article  PubMed  CAS  Google Scholar 

  11. Takise, A., Kodama, A., Shimosato, Y., Watanabe, S., and Suemasu, K. (1988) Histopathologic prognostic factors in adenocarcinoma of the lung periphery less than 2 cm in diameter. Cancer 61, 2083–2088.

    Article  PubMed  CAS  Google Scholar 

  12. Pairolero P. C., Williams D. E., Bergstralh E. J., Piehler J. M., Bernatz P. E., and Payne W. S. (1984) Postsurgical stage I bronchogenic carcinoma: morbid implications of recurrent disease. Ann. Thorac. Surg. 38, 331–336.

    Article  PubMed  CAS  Google Scholar 

  13. Harpole, D. H., Jr., Richards, W. G., Herndon, J. E., and Sugarbaker, D. J. (1996) Angiogenesis and molecular biologic sub-staging in patients with stage I non-small cell lung cancer. Ann. Thorac. Surg. 61, 1470–1476.

    Article  PubMed  Google Scholar 

  14. D’Amico, T. A., Massey, M., Herndon, J. E., II, Moore, M. B., and Harpole, D. H., Jr. (1999) A Biologic risk model for Stage I lung cancer: immunohistochemical analysis of 408 patients using 10 molecular markers. J. Thorac. Cardiovasc. Surg. 117, 736–743.

    Article  PubMed  Google Scholar 

  15. Lau, C. L., D’Amico, T. A., and Harpole, D. H. (1999) Staging and prognosis: clinical and molecular prognostic factors and modes for non-small cell lung cancer, in Lung Cancer: Principles and Practice, 2nd ed. (Pass, H. I., Mitchell, J. B., Johnson, D. H., Turrisi, A. T., and Minna, J. D., eds.), Lippincott, Williams and Wilkins, Philadelphia, PA, pp. 602–611.

    Google Scholar 

  16. Rusch, V. W. and Dmitrovsky, E. (1995) Molecular biologic features of non-small cell lung cancer: clinical implications. Chest Surg. Clin. North Am. 5, 39.

    CAS  Google Scholar 

  17. Slebos, R. J. C., Kibbelaar, R., Dalesio, O., et al. (1990) K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N. Engl. J. Med. 323, 561–565.

    Article  PubMed  CAS  Google Scholar 

  18. Pfeiffer, P., Nexo, E., and Bentzen, S. M. (1998) Enzyme-linked immunosorbent assay of epidermal growth factor receptor in lung cancer: comparisons with immunohistochemistry, clinicopathological features and prognosis. Br. J. Cancer 78, 96–99.

    Article  PubMed  CAS  Google Scholar 

  19. Fujino, S., Enokibori, T., Tezuka, N., et al. (1996) A comparison of epidermal growth factor receptor levels and other prognostic parameters in non-small cell lung cancer. Eur. J. Cancer 32A, 2070–2074.

    Article  PubMed  CAS  Google Scholar 

  20. Greatens, T. M., Niehans, G. A., Rubins, J. B., et al. (1998) Do molecular markers predict survival in non-small cell lung cancer? Am. J. Respir. Crit. Care Med. 157, 1093–1097.

    Article  PubMed  CAS  Google Scholar 

  21. Pastorino, U., Andreola, S., Tagliabue, E., et al. (1997) Immunohistocehmical markers in stage I lung cancer: relevance to prognosis. J. Clin. Oncol. 15, 2858.

    PubMed  CAS  Google Scholar 

  22. Harpole, D. H., Jr., Marks, J. R., et al. (1995b) Localized adenocarcinoma of the lung: oncogene expression of erbB-2 and p53 in 150 patients. Clin. Cancer Res. 1, 659–664.

    PubMed  Google Scholar 

  23. Kern, J. A., Schwartz, D. A., Nordberg, J. E., et al. (1990) P185neu expression in human lung adenocarcinoma predicts shortened survival. Cancer Res. 50, 5184–5187.

    PubMed  CAS  Google Scholar 

  24. Tateishi, M., Ishida, T., Mitsudomi, T., et al. (1991) Prognostic value of c-erbB-2 protein expression in human lung adenocarcinoma and squamous cell carcinoma. Eur. J. Cancer 27, 1372–1375.

    Article  PubMed  CAS  Google Scholar 

  25. Strauss, G. M. (1997) Prognostic markers in resectable non-small cell lung cancer. Hematol. Oncol. Clin. North Am. 11(3), 409–434.

    Article  PubMed  CAS  Google Scholar 

  26. Quinlan, D. C., Davidson, A. G., Summers, C. L., et al. (1992) Accumulation of p53 protein correlates with a poor prognosis in human lung cancer. Cancer Res. 52, 4828–4831.

    PubMed  CAS  Google Scholar 

  27. Fujino, M., Dosaka-Akita, H., Harada, M., et al. (1995) Prognostic significance of p53 and ras p21 expression in non-small cell lung cancer. Cancer 76, 2457–2463.

    Article  PubMed  CAS  Google Scholar 

  28. Kwiatkowski, D. J., Harpole, D. H., Godleski, J., et al. (1998) Molecular pathologic substaging in 244 stage I non-small cell lung cancer patients: clinical implications. J. Clin. Oncol. 16, 2468–2477.

    PubMed  CAS  Google Scholar 

  29. D’Amico, T. A., Aloia, T. A., Moore, M. B., Herndon, J. E., II, Brooks, K. R., Lau, C. L., and Harpole, D. H., Jr. (2000) Molecular biologic substaging of stage I lung cancer according to gender and histology. Ann. Thorac. Surg. 69, 882–886.

    Article  PubMed  Google Scholar 

  30. Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R. D., and Korsmeyer, S. J. (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348, 334–336.

    Article  PubMed  CAS  Google Scholar 

  31. Korsmeyer, S. J. (1992) Bcl-2 initiates a new category of oncogenes: regulators of cell death. Blood 80, 879–886.

    PubMed  CAS  Google Scholar 

  32. Pezzella, F., Turley, H., Duzu, I., et al. (1993) bcl-2 protein in non-small cell lung carcinoma. N. Engl. J. Med. 329, 690–694.

    Article  PubMed  CAS  Google Scholar 

  33. Fontanini, G., Vignati, S., Bigini, D., et al. (1995) Bcl-2 protein: a prognostic factor inversely correlated to p53 in non-small cell lung cancer. Br. J. Cancer 71, 1003–1007.

    Article  PubMed  CAS  Google Scholar 

  34. Wang, T., Szekely, L., Okan, I., Klein, G., and Wiman, K. G. (1993) Wild-type p53-triggered apoptosis is inhibited by bcl-2 in a v-myc-induced T-cell lymphoma line. Oncogene 8, 3427–3431.

    PubMed  CAS  Google Scholar 

  35. Xu, H. J., Quinlan, D. C., Davidson, A. G., et al. (1994) Altered retinoblastoma protein expression and prognosis in early-stage non-small cell lung carcinoma. J. Natl. Cancer Inst. 86, 695–699.

    Article  PubMed  CAS  Google Scholar 

  36. Kratzke, R. A., Greatens, T. M., Rubins, J. B., et al. (1996) Rb and p16INK4a expression in resected non-small cell lung tumors. Cancer Res. 56, 3415–3420.

    PubMed  CAS  Google Scholar 

  37. Pence, J. C., Kerns, B. M., Dodge, R. K., et al. (1993) Prognostic significance of the proliferation index in surgically-resected non-small cell lung cancer. Arch. Surg. 128, 1382–1390.

    Article  PubMed  CAS  Google Scholar 

  38. Macchiarini, P., Fontanini, G., Hardin, M. J., et al. (1992) Relation of neovascularisation to metastasis of non-small cell lung cancer. Lancet 340, 145–146.

    Article  PubMed  CAS  Google Scholar 

  39. Shi, S.-R., Key, M. E., and Kalra, K. L. (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 39, 741–748.

    Article  PubMed  CAS  Google Scholar 

  40. Brandtzaeg, P. and Rognum, T. O. (1984) Evaluation of nine different fixatives: I. Preservation of immunoglobulin isotypes, J chain and secretory components in human tissues. Pathol. Res. Pract. 179, 250–266.

    Article  PubMed  CAS  Google Scholar 

  41. DeLellis, R. A. and Kwan, P. (1988) Technical considerations in the immunohistochemical demonstration of intermediate filaments. Am. J. Surg. Pathol. 12(Suppl. 1), 17–23.

    PubMed  Google Scholar 

  42. Elias, J. (1990a) Immunohistopathology: A Practical Approach to Diagnosis. ASCP Press, Chicago, IL.

    Google Scholar 

  43. Werner, M., Von Wasielewski, R., and Komminoth, P. (1996) Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem. Cell Biol. 105(4), 53–60.

    Article  Google Scholar 

  44. Antel, J. P., Kuchibhotla, J., and Stefansson, K. (1985) Generation of monoclonal antibodies recognizing neuronal elements in formalin-fixed, paraffin-embedded human tissue. J. Neuropathol. Exp. Neurol. 44, 533–545.

    Article  PubMed  CAS  Google Scholar 

  45. West, K. P., Warford, A., Fray, L., et al. (1986) The demonstration of B cell, T cell and myeloid antigens in paraffin sections. J. Pathol. 150(2), 89–101.

    Article  PubMed  CAS  Google Scholar 

  46. de Vente, J., Steinbusch, H. W. M., and Schipper, J. (1987) A new approach to immunocytochemistry of 3′,5′-cyclic guanosine monophosphate: preparation, specificity, and initial application of a new antiserum against formaldehyde-fixed 3′,5′-cyclic guanosine monophosphate. Neuroscience 22, 361–373.

    Article  PubMed  Google Scholar 

  47. Kaku, T., Ekem, J. K., Lindayen, C., et al. (1983) Comparison of formalin-and acetone-fixation for immunohistochemical detection of carcinoembryonic antigen (CEA) and keratin. Am. J. Clin. Pathol. 80, 806–815.

    PubMed  CAS  Google Scholar 

  48. Reading, M. (1977) A digestion technique for the reduction of background staining in the immunoperoxidase method. J. Clin. Pathol. 30, 88–90.

    Article  PubMed  CAS  Google Scholar 

  49. Curran, R. C. and Gregory, J. (1977) The unmasking of antigens in paraffin sections by trypsin. Experientia 33, 1400–1401.

    Article  PubMed  CAS  Google Scholar 

  50. Jacobsen, M., Clausenper, P., and Schmidth, S. (1980) The effect of fixation and trypsinization of the immunohistochemical demonstration of intracellular immunoglobulin in paraffin embedded material. Acta Pathol. Microbiol. Immunol. Scand. A 88, 368–376.

    Google Scholar 

  51. Elias, J. M. (1990b) Estrogen receptor localization in paraffin sections using enzyme digestion, repeated applications of primary antibody, and imidazole. J. Histotechnol. 13, 29–33.

    CAS  Google Scholar 

  52. Heyderman, E. (1979) Immunoperoxidase technique in histopathology: applications, methods, and controls. J. Clin. Pathol. 32(10), 971–978.

    Article  PubMed  CAS  Google Scholar 

  53. Battifora, H. (1991) Assessment of antigen damage in immunohistochemistry. Am. J. Clin. Pathol. 96, 669–671.

    PubMed  CAS  Google Scholar 

  54. Ordonez, N. G., Manning, J. T., and Brooks, T. E. (1988) Effect of trypsination on the immunostaining of formalin-fixed, paraffin-embedded tissues. Am. J. Surg. Pathol. 12, 121–129.

    Article  PubMed  CAS  Google Scholar 

  55. Taylor, C. R, Shi, S. R., Chaiwum, B., Young, L., Imam, A., and Cote, R. J. (1994) Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections. Hum. Pathol. 25, 163–270.

    Google Scholar 

  56. Fraenkel-Conrat, H. and Olcott, H. A. Reaction of formaldehyde with proteins. (1947) VI Cross-linking of amino groups with phenol, imidazole, or indole groups. J. Biol. Chem. 174, 827–843.

    Google Scholar 

  57. Fraenkel-Conrat, H., Brandon, B. A., and Olcott, H. A. (1948) The reaction of formaldehyde with proteins. IV Participation of indole groups. Gramicidin. J. Biol. Chem. 168, 99–118.

    Google Scholar 

  58. Morgan, J. M., Navabi, H., Schmid, K. W., and Jasani, B. (1994) Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J. Pathol. 147, 301–307.

    Article  Google Scholar 

  59. Cattoretti, G. and Suurmeijer, A. J. H. (1995) Antigen unmasking on formalin-fixed, paraffin-embedded tissues using microwaves: a review. Adv. Anat. Pathol. 2, 2–9.

    Article  Google Scholar 

  60. Hsu, S. M., Raine, L., and Fanger, H. (1981a) A comparitive study of the peroxidase-anti-peroxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am. J. Clin. Pathol. 75(5), 734–738.

    PubMed  CAS  Google Scholar 

  61. Hsu, S. M., Raine, L., and Fanger, H. (1981b) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabled antibody (PAP) procedures. J. Histochem. Cytochem. 29(4), 577–580.

    Article  PubMed  CAS  Google Scholar 

  62. Chaiet, L. and Wolf, F. J. (1964) The properties of streptavidin a biotin-bonding protein produced by streptomyces. Arch. Biochem. Biophys. 106, 1–5.

    Article  PubMed  CAS  Google Scholar 

  63. Kuhlmann, W. and Krischan, R. (1981) Resin embedment of organs and postembeddment localization of antigens by immunoperoxidase methods. Histochemistry 72, 377–389.

    Article  PubMed  CAS  Google Scholar 

  64. Naritoku, W. Y. and Taylor, C. R. (1982) A comparative study of the use of monoclonal antibodies using three different immunohistochemical methods: an evaluation of monoclonal and polyclonal antibodies against human prostatic acid phosphatase. J. Histochem. Cytochem. 30, 253–260.

    Article  PubMed  CAS  Google Scholar 

  65. Pedraza, M. A., Mason, D., Doslu, R. A., Marsh, R. A., and Boblett, J. P. (1984) Immmunoperoxidase methods with plastic embedded materials. Lab. Med. 15(2), 113–115.

    CAS  Google Scholar 

  66. Woods, G. S. and Warnke, R. (1981) Suppression of endogenous avidin-binding activity in tissues and its relevance to biotin-avidin detection system. J. Histochem. Cytochem. 29(10), 1196–1204.

    Article  Google Scholar 

  67. Taylor, C. R. (2000) The total test approach to standardization of immunohisto-chemistry. Arch. Pathol. Lab. Med. 124, 945–951.

    PubMed  CAS  Google Scholar 

  68. Tacha, D. E. and Chen, T. (1994) Modified antigen retrieval procedure calibration technique for microwave ovens. J. Histotechnol. 17(4), 365–366.

    Article  Google Scholar 

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Joshi, MB.M., D’Amico, T.A., Harpole, D.H. (2003). Molecular Biologic Substaging of Stage I NSCLC Through Immunohistochemistry Performed on Formalin-Fixed, Paraffin-Embedded Tissue. In: Driscoll, B. (eds) Lung Cancer. Methods in Molecular Medicine™, vol 75. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-324-0:369

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  • DOI: https://doi.org/10.1385/1-59259-324-0:369

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-0-89603-920-9

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