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Does the evaluation of proliferative activity predict malignancy or prognosis in endocrine tumors?
Human PATHOLOGY VOLUME 26
February 1995
NUMBER 2
Editorial Does the Evaluation of Proliferative Activity Predict Malignancy or Prognosis in Endocrine Tumors? Evaluation of the malignant potential of endocrine tumors in the absence of overt invasion or metastasis often presents a formidable challenge to the surgical pathologist. In some instances, as in the case of adrenal cortical tumors, specific features, including tumor weight, necrosis, fibrous bands, nuclear atypia, mitotic activity, and selected clinical parameters, have been used successfully in multiparametric analyses to predict biological behavior. *-3 In other instances patterns of h o r m o n e expression have b e c o m e important adjunctive criteria for prediction of malignancy. Pancreatic endocrine tumors producing gastrin or somatostatin, for example, are far more likely to be malignant than those tumors producing insulin. ~ For many other endocrine tumors criteria for malignancy either have not been adequately described or have not been applied to large e n o u g h series of cases to assess their validity. Estimation of proliferative activity has emerged as a major approach for the determination of the aggressiveness, progression, and metastatic potential of a variety of h u m a n neoplasms. Although this feature has been studied most extensively in tumors by flow cytometry, numerous other approaches have been developed for the analysis of proliferative activity. 5 These have ranged from the use of radioactively labeled (thymidine) or nonlabeled (bromodeoxyuridine) DNA precursors for S-phase analysis to the use of monoclonal antibodies to various components of proliferating cells. Each of these methods, however, measures a different parameter of proliferative activity. The issue of mitotic counting in tumors has been discussed extensively in the pages of this journal. 6-1° Although estimation of mitotic rate intuitively seems to be the most direct and simple m e t h o d for estimating tumor cell proliferation, this approach is fraught with difficulty. It should be r e m e m b e r e d that the M phase of the mitotic cycle is quite short and that the numbers
131
of mitoses might not accurately reflect the overall proliferative activity of any particular tumor. 5 Moreover, sections that are poorly fixed, thickly sectioned, or overstained may present considerable interpretative difficulties. As a result, levels of reproducibility for estimation of mitotic rates have been poor. An additional problem results from delays in fixation. Donhuijsen et al 6 measured a 13% decrease in the n u m b e r of recognizable mitoses after a 3-hour delay in fixation and a 46% decrease after a 12-hour delay. Much of this decrease stems from a reduced identifiability of mitotic figures resulting from autolytic changes, which make the distinction of mitoses and pyknotic nuclei impossible. Additionally, some of the decreases in mitotic rate related to fixation delays also result from completion of mitotic cycles. Another approach to the study of proliferative activity is the use of monoclonal antibodies to various components of proliferating cells. ~Although many such antibodies have been developed over the years, the most commonly used are those directed to proliferating cell nuclear antigen (PCNA),n-13 Ki_67,14,a5 and the formalin resistant analogue of Ki-67 (MIB-1).16 Proliferating cell nuclear antigen is a 36-kd acidic nuclear protein that is essential for DNA synthesis and is present in 19 highest concentrations during the S phase. It appears late in the G1 phase and its levels progressively decrease in the G2 and M phases. Although PCNA immunoreactivity can be demonstrated effectively both in methanoland formalin-fixed tissues, prolonged formalin fixation interferes substantially with staining. 17'18The MIB-1 antibody reacts in formalin-fixed, paraffin-embedded tissue, after microwave antigen retrieval, with a proliferation associated antigen that constitutes a portion of the Ki-67 molecule. 14 The expression of this marker begins in the G1 phase and progressively increases through the M phase; it is absent from cells in the GO phase. ~'16 Weidner et al 8 showed a strong correlation between the
HUMAN PATHOLOGY
Volume 26, No. 2 (February 1995)
Ki-67 labeling index (MIB-l-positive cells/I,000 cells) and the mitotic figure index (numbers of mitoses/1,000 cells) in a series of breast carcinomas. T h e r e is a growing body of literature to support the view that proliferation markers, such as Ki-67, can provide useful prognostic information for a variety of different neoplasms. 15 For example, Weidner et al showed a strong correlation between the Ki-67 labeling indices and the Scraff-Bloom-Richardson grades of breast carcinomas, whereas other investigators have shown a significant association between the extent of Ki-67 labelin~ and size of the primary tumor and axillary node status?" Up to the present time, however, there have been relatively few studies of the utility of this approach in endocrine tumors. Goldblum et al 2° studied a series of adrenal cortical tumors and showed that Ki-67 scores and labeling indices were significantly higher in carcinomas than in adenomas and that these parameters correlated with mitotic counts and clinical outcome. Pelosi et a121 employed PCNA antibodies in a series of functioning and nonfunctioning pancreatic endocrine tumors. Tumors with a PCNA index greater than 5%, including both functioning and nonfunctioning tumors, had extrapancreatic extension. In a series of thyroid tumors Carr et a122 showed the highest proportion of Ki-67-positive cells in anaplastic carcinomas (20.4%), whereas the values in medullary, papillary, and follicular carcinomas were 4.3%, 3.6%, and 0.5%, respectively. Moreover, there was a significant difference in labeling indices in papillary carcinomas with or without extrathyroidal extension. Kitz et al 2~ studied the distribution of Ki-67 in a large series of pituitary adenomas and showed that tumors with histologically proven dural infiltration had a statistically significant higher Ki67 labeling index than did tumors without this feature. In the parathyroid Loda et a124 showed that nodular foci, which are found commonly in adenomas and hyperplasias, contain subpopulations of cells with consistently higher rates of labeling with PCNA and MIB-1 than diffuse areas. They suggested that cells within such nodules may be more likely to develop the genetic abnormalities that have been observed both in adenomas and hyperplasias. 25 Two articles in this issue of HUMAN PATHOLOGY explore the feasibility of using proliferation markers to assess the malignant potential of parathyroid 26 and thyroid 27 tumors. The difficulty of distinguishing parathyroid carcinomas and adenomas is well known. The classical study of parathyroid carcinomas by Schantz a nd Ca stleman 28 suggested that the presence of parenchymal mitoses was the single most important criterion for the diagnosis of carcinoma. Mitoses were present in 81% of their carcinomas; however, three of 10 patients with no mitoses in their tumors had recurrent or metastatic tumors or died of their disease, whereas two of three patients whose tumors contained many mitoses were alive and well. Subsequent studies have shown that mitoses may be present in benign lesions of the parathyroid, including adenomas and hyperplasias. Snover and Foucar, 29 for example, found mitoses in 71% of adenomas. Fifty-nine percent of their tumors contained fewer
132
than 1 mitosis/10 high power fields, whereas 12% had more than 1 mitosis/10 high power fields. These data indicate that occasional mitotic activity, by itself, cannot be considered a criterion of malignancy in the parathyroid. In a recent study of parathyroid carcinomas selected on the basis of proven invasive growth, local recurrences, and distant metastases, Bondeson et al ~° showed that mitotic activity is of limited diagnostic significance for the distinction of clinically benign parathyroid lesions and carcinomas. In half of the carcinomas the mitotic rate did not exceed that obtained in clinically benign parathyroid tumors. However, mitotic activity did provide an important prognostic parameter, a° Mitotic rates in excess of 5 / 5 0 high power fields, macronucleoli, and necrosis were associated with an aggressive behavior with respect to the development of recurrent or metastatic disease. Most recently, staining for the retinoblastoma (Rb) protein has been found to be useful for the distinction of parathyroid carcinomas that are Rb negative and adenomas that are Rb positive. 31 Whether the absence of Rb relates to clinical aggression, however, is unknown. In their study of parathyroid tumors Abbona et a126 conclude that proliferative activity, as determined by staining with MIB-1, is a useful parameter for the distinction of parathyroid carcinomas and adenomas. T h e r e was a statistically significant difference (P < .05) in MIB-1 scores between the entire group of carcinomas and parathyroid adenomas or hyperplasias. T h e r e were no significant differences, however, between nonaggressive carcinomas and adenomas. On the other hand, both mitotic counts and MIB-1 scores of clinically aggressive carcinomas were significantly different from those of adenomas (P < .01). These results thus parallel observations made by Bondeson et al with respect to the prognostic significance of the high mitotic counts of clinically aggressive carcinomas. 30 Although MIB-1 counts were not useful for the distinction of adenomas and nonaggressive carcinomas, they did provide important prognostic information with respect to predicting clinical aggression. Katoh et a127 studied a wide spectrum of hyperpiastic and neoplastic lesions of the thyroid also using the MIB-1 proliferation marker. As expected, undifferentiated thyroid carcinomas had the highest proportion of labeled nuclei, whereas papillary carcinomas had the lowest. Although there was a statistically significant difference (P < .01) between the extent of labeling of papillary carcinomas and the entire group of follicular carcinomas, there were no differences between minimally invasive follicular and papillary carcinomas. Statistically significant differences, however, were apparent between widely and minimally invasive follicular carcinomas. Thus, the results of these studies confirm what we would expect. The high proliferative activity of undifferentiated carcinomas parallels their highly aggressive course, whereas the low proliferative activity of papillary and minimally invasive follicular carcinomas (differentiated thyroid carcinomas) reflects their usually indolent behavior. Additionally, widely invasive fol•
EDITORIAL (Ronald A. DeLellis)
licular carcinomas result in a worse prognosis than their minimally invasive counterparts. With respect to benign lesions, although there were significant differences between a d e n o m a and adenomatous goiter, these differences disappeared with the exclusion of the solidtrabecular adenomas, which had a relatively high proliferative rate. Similarly, there were no differences between minimally invasive follicular carcinomas and follicular adenomas. MIB-1 offers substantial benefits over traditional methods of mitotic counting. In most instances formalin-fixed, paraffin-embedded sections can be used provided that appropriate microwave retrieval steps have been p e r f o r m e d beforehand. Moreover, the MIB-1 sections also can be subjected to quantitative analysis using image analysis systems. 32 As demonstrated in the two studies reported in this issue, M1B-1 can be used to discriminate broad sets of thyroid and parathyroid tumors into aggressive and nonaggressive categories. However, because of the overlap of values for individual tumors, areas of particular diagnostic difficulty (parathyroid a d e n o m a v nonaggressive parathyroid carcin o m a or minimally invasive follicular thyroid carcinoma v follicular adenoma) do not appear to benefit from this approach. Multiparametric approaches using additional markers as well as traditional histopathological features may help to resolve these dilemmas. For example, could the evaluation of the Rb protein by immunohistochemistry, as r e p o r t e d by Cryns et al, 31 further refine the distinction of parathyroid adenomas and carcinomas when c o m b i n e d with MIB-1 analysis? Similarly, could the addition o f t u m o r suppressor and oncogene analyses, together with MIB-1 counts, refine the classification o f thyroid neoplasms? 22'33 T h e answers to these questions u n d o u b t e d l y will have to await f u r t h e r investigation. For the present time MIB-1 by itself c a n n o t be used to j u d g e the malignancy of individual e n d o c r i n e tumors, but this marker may be helpful in identifying subsets of tumors that will have an aggressive course. RONALD A. DELELLIS, M D Department of Pathology New England Medical Center 7 5 0 W a s h i n g t o n St
Boston, MA 02111
REFERENCES 1. van Slooten H, Schaberg A, Smeenk D, et al: Morphological characteristics of benign and malignant adrenal cortical tumors. Cancer 55:766-773, 1985 2. Weiss LM: Comparative histological study of 43 metastasizing and non-metastasizing adrenal cortical tumors. Am J Surg Pathol 8:163-169, 1984 3. Hough A, Hollifield fW, Page DL, et al: Prognostic factors in adrenal cortical tumors. A mathematical analysis of clinical and morphological data. Am J Clin Pathol 72:390-399, 1979 4. Heitz PU: Pancreatic endocrine tumors, in K16ppel G, Heitz PU (eds): Pancreatic Pathology. Edinburgh, Scotland, Churchill Livingstone, 1984, pp 206-232
133
5. Weinberg DS: Proliferation indices in solid tumors. Adv Pathol Lab Med 5:163-191, 1993 6. Donhuijsen K, Schmidt U, Hirche H, et al: Mitosis counts: Reproducibility and significance in grading malignancy. HUM PATHOL 17:1122-1125, 1986 7. BaakJPA: Mitosis counting in tumors. HUM PATHOL 21:683685, 1990 8. Weidner N, Moore DH, Vartanian R: Correlation of Ki-67 antigen expression with mitotic figure index and tumor grade in breast carcinomas using the novel paraffin reaction MIB-1 antibody. HUM PATHOL 25:337-342, 1994 9. SimpsonJF, Page DL: Cellular proliferation and prognosis in breast cancer: Statistical purity versus clinical utility. HUM PATHOL 25:331-332, 1994 10. Going JJ: Efficiently estimated histologic cell counts. HUM PATHOL 25:333-336, 1994 11. Ogata K, Kurki P, Celis JE, et al: Monoclonal antibodies to nuclear protein (PCNA/cyclin) associated with DNA replication. Exp Cell Res 168:475-486, 1987 12. Bravo R, Frank R, Blundell PA, et al: Cyclin/PCNA is the auxiliary protein of DNA polymerase delta. Nature 326:515-520, 1987 13. Kamel OW, LeBrun DP, Davis RE, et al: Growth fraction estimation of malignant lymphomas in formalin-fixed, paraffin-embedded tissues using anti-PCNA/cyclin 19A2. Correlation with Ki-67 labelling. Am J Pathol 138:1471-1476, 1991 14. GerdesJ, Lemke H, Baisch H, et al: Cell cycle analysis of a cell proliferation associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710-1715, 1984 15. Brown DC, Gatter KC: Monoclonal antibody Ki-67: Its use in histopathology. Histopathology 17:489-503, 1990 16. Cattoretti G, Becker MHG, Key G, et al: Monoclonal antibodies against r e c o m b i n a n t parts of the Ki-67 antigen (MIB-1 and MIB-3) detect proliferating cells in microwave processed formalin-fixed paraffin-embedded sections. J Pathol 168:357-363, 1992 17. Cohrera MD, SkellyM, Gown AM: Anti-PCNA antibody PCl0 yields unreliable proliferation indices in routinely processed, deparaffinized, formalin-fixed tissue. Appl Immunohistochem 1:193-200, 1993 18. Burford-Mason AP, MacKay AJ, Cummins M, et al: Detection of proliferating cell nuclear antigen in paraffin-embedded specimens is dependent on pre-embedding tissue handling and fixation. Arch Pathol Lab Med 118:100%1013, 1994 19. Wintzer H-O, Zipfel I, Schulte-MontingJ, et al: Ki-67 immunostaining in human breast tumors and its relationship to prognosis. Cancer 67:421-428, 1991 20. GoldblumJR, Shannon R, Kaldijian EP, et al: Immunohistochemical assessment of proliferative activity in adrenocortical neoplasms. Mod Pathol 6:663-668, 1993 21. Pelosi G, Zamboni G, Doglioni C, et al: Immunodetection of proliferating cell nuclear antigen assesses the growth fraction and predicts malignancy in endocrine rumors of the pancreas. Am J Surg Pathol 16:1215-1225, 1992 22. Carr K, Heffess C,Jin L, et al: Immunohistochemical analysis of thyroid carcinomas utilizing antibodies to p53 and Ki-67. Appl Immunohistochem 1:201-207, 1993 23. Kitz K, Knosp E, Koos WT, et al: Proliferation of pituitary adenomas: Measurement by Mab Ki-67. Acta Neurochiurg 53:60-64, 1991 (suppl) 24. Loda M, Lipman J, Cukor B, et al: Nodular foci in parathyroid adenomas and hyperplasias. An immunohistochemical study of proliferative activity. HUM PATHOL 25:1050-1056, 1994 25. Thakker RV: The role of molecular genetics in screening for multiple endocrine neoplasia type 1. Endocrinol Clin North Am 23:117-136, 1994 26. Abbona GC, Papotti M, Gasparri G, et al: Proliferative activity in parathyroid tumors as detected by Ki-67 immunostaining. HUM PATHOL 26:135-138, 1995 27. Katoh R, Bray CE, Suzuki K, et al: Growth activity in hyperplastic and neoplastic human thyroid determined by an immunohistochemical staining procedure using monoclonal antibody MIB-1. HUM PATHOL 26:139-146, 1995
HUMAN PATHOLOGY
Volume 26, No. 2 (February 1995)
28. Schantz A, Castleman B: Parathyroid carcinoma. A study of 70 cases. Cancer 31:600-605, 1973 29. Shover DC, Foucar K: Mitotic activity in benign thyroid diseases. A m J Clin Pathol 75:345-347, 1981 30. Bondeson L, Sandelin K, Grimelius L: Histopathologic variables and DNA cytometry in parathyroid carcinoma. AmJ Surg Pathol 17:820-829, 1993 31. Cryns VL, Thor A, Xu H-J, et ah Loss of the retinoblastoma
134
tumor suppressor gene in parathyroid carcinoma. New Engl J Med 330:757-761, 1994 32. Herzberg AJ, Kerns BJ, Honkanen FA, et al: DNA ploidy and proliferation index of soft tissue sarcomas determined by image cytometry of fresh frozen tissue. Am J Clin Pathol 97:$29-$37, 1992 (suppl) 33. Dobashi Y, Sugimura H, Sakamoto A: Step wise participation of p53 gene mutation during dedifferentiation of human thyroid carcinomas. Diagn Mol Pathol 3:9-14, 1994
February 1995
NUMBER 2
Editorial Does the Evaluation of Proliferative Activity Predict Malignancy or Prognosis in Endocrine Tumors? Evaluation of the malignant potential of endocrine tumors in the absence of overt invasion or metastasis often presents a formidable challenge to the surgical pathologist. In some instances, as in the case of adrenal cortical tumors, specific features, including tumor weight, necrosis, fibrous bands, nuclear atypia, mitotic activity, and selected clinical parameters, have been used successfully in multiparametric analyses to predict biological behavior. *-3 In other instances patterns of h o r m o n e expression have b e c o m e important adjunctive criteria for prediction of malignancy. Pancreatic endocrine tumors producing gastrin or somatostatin, for example, are far more likely to be malignant than those tumors producing insulin. ~ For many other endocrine tumors criteria for malignancy either have not been adequately described or have not been applied to large e n o u g h series of cases to assess their validity. Estimation of proliferative activity has emerged as a major approach for the determination of the aggressiveness, progression, and metastatic potential of a variety of h u m a n neoplasms. Although this feature has been studied most extensively in tumors by flow cytometry, numerous other approaches have been developed for the analysis of proliferative activity. 5 These have ranged from the use of radioactively labeled (thymidine) or nonlabeled (bromodeoxyuridine) DNA precursors for S-phase analysis to the use of monoclonal antibodies to various components of proliferating cells. Each of these methods, however, measures a different parameter of proliferative activity. The issue of mitotic counting in tumors has been discussed extensively in the pages of this journal. 6-1° Although estimation of mitotic rate intuitively seems to be the most direct and simple m e t h o d for estimating tumor cell proliferation, this approach is fraught with difficulty. It should be r e m e m b e r e d that the M phase of the mitotic cycle is quite short and that the numbers
131
of mitoses might not accurately reflect the overall proliferative activity of any particular tumor. 5 Moreover, sections that are poorly fixed, thickly sectioned, or overstained may present considerable interpretative difficulties. As a result, levels of reproducibility for estimation of mitotic rates have been poor. An additional problem results from delays in fixation. Donhuijsen et al 6 measured a 13% decrease in the n u m b e r of recognizable mitoses after a 3-hour delay in fixation and a 46% decrease after a 12-hour delay. Much of this decrease stems from a reduced identifiability of mitotic figures resulting from autolytic changes, which make the distinction of mitoses and pyknotic nuclei impossible. Additionally, some of the decreases in mitotic rate related to fixation delays also result from completion of mitotic cycles. Another approach to the study of proliferative activity is the use of monoclonal antibodies to various components of proliferating cells. ~Although many such antibodies have been developed over the years, the most commonly used are those directed to proliferating cell nuclear antigen (PCNA),n-13 Ki_67,14,a5 and the formalin resistant analogue of Ki-67 (MIB-1).16 Proliferating cell nuclear antigen is a 36-kd acidic nuclear protein that is essential for DNA synthesis and is present in 19 highest concentrations during the S phase. It appears late in the G1 phase and its levels progressively decrease in the G2 and M phases. Although PCNA immunoreactivity can be demonstrated effectively both in methanoland formalin-fixed tissues, prolonged formalin fixation interferes substantially with staining. 17'18The MIB-1 antibody reacts in formalin-fixed, paraffin-embedded tissue, after microwave antigen retrieval, with a proliferation associated antigen that constitutes a portion of the Ki-67 molecule. 14 The expression of this marker begins in the G1 phase and progressively increases through the M phase; it is absent from cells in the GO phase. ~'16 Weidner et al 8 showed a strong correlation between the
HUMAN PATHOLOGY
Volume 26, No. 2 (February 1995)
Ki-67 labeling index (MIB-l-positive cells/I,000 cells) and the mitotic figure index (numbers of mitoses/1,000 cells) in a series of breast carcinomas. T h e r e is a growing body of literature to support the view that proliferation markers, such as Ki-67, can provide useful prognostic information for a variety of different neoplasms. 15 For example, Weidner et al showed a strong correlation between the Ki-67 labeling indices and the Scraff-Bloom-Richardson grades of breast carcinomas, whereas other investigators have shown a significant association between the extent of Ki-67 labelin~ and size of the primary tumor and axillary node status?" Up to the present time, however, there have been relatively few studies of the utility of this approach in endocrine tumors. Goldblum et al 2° studied a series of adrenal cortical tumors and showed that Ki-67 scores and labeling indices were significantly higher in carcinomas than in adenomas and that these parameters correlated with mitotic counts and clinical outcome. Pelosi et a121 employed PCNA antibodies in a series of functioning and nonfunctioning pancreatic endocrine tumors. Tumors with a PCNA index greater than 5%, including both functioning and nonfunctioning tumors, had extrapancreatic extension. In a series of thyroid tumors Carr et a122 showed the highest proportion of Ki-67-positive cells in anaplastic carcinomas (20.4%), whereas the values in medullary, papillary, and follicular carcinomas were 4.3%, 3.6%, and 0.5%, respectively. Moreover, there was a significant difference in labeling indices in papillary carcinomas with or without extrathyroidal extension. Kitz et al 2~ studied the distribution of Ki-67 in a large series of pituitary adenomas and showed that tumors with histologically proven dural infiltration had a statistically significant higher Ki67 labeling index than did tumors without this feature. In the parathyroid Loda et a124 showed that nodular foci, which are found commonly in adenomas and hyperplasias, contain subpopulations of cells with consistently higher rates of labeling with PCNA and MIB-1 than diffuse areas. They suggested that cells within such nodules may be more likely to develop the genetic abnormalities that have been observed both in adenomas and hyperplasias. 25 Two articles in this issue of HUMAN PATHOLOGY explore the feasibility of using proliferation markers to assess the malignant potential of parathyroid 26 and thyroid 27 tumors. The difficulty of distinguishing parathyroid carcinomas and adenomas is well known. The classical study of parathyroid carcinomas by Schantz a nd Ca stleman 28 suggested that the presence of parenchymal mitoses was the single most important criterion for the diagnosis of carcinoma. Mitoses were present in 81% of their carcinomas; however, three of 10 patients with no mitoses in their tumors had recurrent or metastatic tumors or died of their disease, whereas two of three patients whose tumors contained many mitoses were alive and well. Subsequent studies have shown that mitoses may be present in benign lesions of the parathyroid, including adenomas and hyperplasias. Snover and Foucar, 29 for example, found mitoses in 71% of adenomas. Fifty-nine percent of their tumors contained fewer
132
than 1 mitosis/10 high power fields, whereas 12% had more than 1 mitosis/10 high power fields. These data indicate that occasional mitotic activity, by itself, cannot be considered a criterion of malignancy in the parathyroid. In a recent study of parathyroid carcinomas selected on the basis of proven invasive growth, local recurrences, and distant metastases, Bondeson et al ~° showed that mitotic activity is of limited diagnostic significance for the distinction of clinically benign parathyroid lesions and carcinomas. In half of the carcinomas the mitotic rate did not exceed that obtained in clinically benign parathyroid tumors. However, mitotic activity did provide an important prognostic parameter, a° Mitotic rates in excess of 5 / 5 0 high power fields, macronucleoli, and necrosis were associated with an aggressive behavior with respect to the development of recurrent or metastatic disease. Most recently, staining for the retinoblastoma (Rb) protein has been found to be useful for the distinction of parathyroid carcinomas that are Rb negative and adenomas that are Rb positive. 31 Whether the absence of Rb relates to clinical aggression, however, is unknown. In their study of parathyroid tumors Abbona et a126 conclude that proliferative activity, as determined by staining with MIB-1, is a useful parameter for the distinction of parathyroid carcinomas and adenomas. T h e r e was a statistically significant difference (P < .05) in MIB-1 scores between the entire group of carcinomas and parathyroid adenomas or hyperplasias. T h e r e were no significant differences, however, between nonaggressive carcinomas and adenomas. On the other hand, both mitotic counts and MIB-1 scores of clinically aggressive carcinomas were significantly different from those of adenomas (P < .01). These results thus parallel observations made by Bondeson et al with respect to the prognostic significance of the high mitotic counts of clinically aggressive carcinomas. 30 Although MIB-1 counts were not useful for the distinction of adenomas and nonaggressive carcinomas, they did provide important prognostic information with respect to predicting clinical aggression. Katoh et a127 studied a wide spectrum of hyperpiastic and neoplastic lesions of the thyroid also using the MIB-1 proliferation marker. As expected, undifferentiated thyroid carcinomas had the highest proportion of labeled nuclei, whereas papillary carcinomas had the lowest. Although there was a statistically significant difference (P < .01) between the extent of labeling of papillary carcinomas and the entire group of follicular carcinomas, there were no differences between minimally invasive follicular and papillary carcinomas. Statistically significant differences, however, were apparent between widely and minimally invasive follicular carcinomas. Thus, the results of these studies confirm what we would expect. The high proliferative activity of undifferentiated carcinomas parallels their highly aggressive course, whereas the low proliferative activity of papillary and minimally invasive follicular carcinomas (differentiated thyroid carcinomas) reflects their usually indolent behavior. Additionally, widely invasive fol•
EDITORIAL (Ronald A. DeLellis)
licular carcinomas result in a worse prognosis than their minimally invasive counterparts. With respect to benign lesions, although there were significant differences between a d e n o m a and adenomatous goiter, these differences disappeared with the exclusion of the solidtrabecular adenomas, which had a relatively high proliferative rate. Similarly, there were no differences between minimally invasive follicular carcinomas and follicular adenomas. MIB-1 offers substantial benefits over traditional methods of mitotic counting. In most instances formalin-fixed, paraffin-embedded sections can be used provided that appropriate microwave retrieval steps have been p e r f o r m e d beforehand. Moreover, the MIB-1 sections also can be subjected to quantitative analysis using image analysis systems. 32 As demonstrated in the two studies reported in this issue, M1B-1 can be used to discriminate broad sets of thyroid and parathyroid tumors into aggressive and nonaggressive categories. However, because of the overlap of values for individual tumors, areas of particular diagnostic difficulty (parathyroid a d e n o m a v nonaggressive parathyroid carcin o m a or minimally invasive follicular thyroid carcinoma v follicular adenoma) do not appear to benefit from this approach. Multiparametric approaches using additional markers as well as traditional histopathological features may help to resolve these dilemmas. For example, could the evaluation of the Rb protein by immunohistochemistry, as r e p o r t e d by Cryns et al, 31 further refine the distinction of parathyroid adenomas and carcinomas when c o m b i n e d with MIB-1 analysis? Similarly, could the addition o f t u m o r suppressor and oncogene analyses, together with MIB-1 counts, refine the classification o f thyroid neoplasms? 22'33 T h e answers to these questions u n d o u b t e d l y will have to await f u r t h e r investigation. For the present time MIB-1 by itself c a n n o t be used to j u d g e the malignancy of individual e n d o c r i n e tumors, but this marker may be helpful in identifying subsets of tumors that will have an aggressive course. RONALD A. DELELLIS, M D Department of Pathology New England Medical Center 7 5 0 W a s h i n g t o n St
Boston, MA 02111
REFERENCES 1. van Slooten H, Schaberg A, Smeenk D, et al: Morphological characteristics of benign and malignant adrenal cortical tumors. Cancer 55:766-773, 1985 2. Weiss LM: Comparative histological study of 43 metastasizing and non-metastasizing adrenal cortical tumors. Am J Surg Pathol 8:163-169, 1984 3. Hough A, Hollifield fW, Page DL, et al: Prognostic factors in adrenal cortical tumors. A mathematical analysis of clinical and morphological data. Am J Clin Pathol 72:390-399, 1979 4. Heitz PU: Pancreatic endocrine tumors, in K16ppel G, Heitz PU (eds): Pancreatic Pathology. Edinburgh, Scotland, Churchill Livingstone, 1984, pp 206-232
133
5. Weinberg DS: Proliferation indices in solid tumors. Adv Pathol Lab Med 5:163-191, 1993 6. Donhuijsen K, Schmidt U, Hirche H, et al: Mitosis counts: Reproducibility and significance in grading malignancy. HUM PATHOL 17:1122-1125, 1986 7. BaakJPA: Mitosis counting in tumors. HUM PATHOL 21:683685, 1990 8. Weidner N, Moore DH, Vartanian R: Correlation of Ki-67 antigen expression with mitotic figure index and tumor grade in breast carcinomas using the novel paraffin reaction MIB-1 antibody. HUM PATHOL 25:337-342, 1994 9. SimpsonJF, Page DL: Cellular proliferation and prognosis in breast cancer: Statistical purity versus clinical utility. HUM PATHOL 25:331-332, 1994 10. Going JJ: Efficiently estimated histologic cell counts. HUM PATHOL 25:333-336, 1994 11. Ogata K, Kurki P, Celis JE, et al: Monoclonal antibodies to nuclear protein (PCNA/cyclin) associated with DNA replication. Exp Cell Res 168:475-486, 1987 12. Bravo R, Frank R, Blundell PA, et al: Cyclin/PCNA is the auxiliary protein of DNA polymerase delta. Nature 326:515-520, 1987 13. Kamel OW, LeBrun DP, Davis RE, et al: Growth fraction estimation of malignant lymphomas in formalin-fixed, paraffin-embedded tissues using anti-PCNA/cyclin 19A2. Correlation with Ki-67 labelling. Am J Pathol 138:1471-1476, 1991 14. GerdesJ, Lemke H, Baisch H, et al: Cell cycle analysis of a cell proliferation associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710-1715, 1984 15. Brown DC, Gatter KC: Monoclonal antibody Ki-67: Its use in histopathology. Histopathology 17:489-503, 1990 16. Cattoretti G, Becker MHG, Key G, et al: Monoclonal antibodies against r e c o m b i n a n t parts of the Ki-67 antigen (MIB-1 and MIB-3) detect proliferating cells in microwave processed formalin-fixed paraffin-embedded sections. J Pathol 168:357-363, 1992 17. Cohrera MD, SkellyM, Gown AM: Anti-PCNA antibody PCl0 yields unreliable proliferation indices in routinely processed, deparaffinized, formalin-fixed tissue. Appl Immunohistochem 1:193-200, 1993 18. Burford-Mason AP, MacKay AJ, Cummins M, et al: Detection of proliferating cell nuclear antigen in paraffin-embedded specimens is dependent on pre-embedding tissue handling and fixation. Arch Pathol Lab Med 118:100%1013, 1994 19. Wintzer H-O, Zipfel I, Schulte-MontingJ, et al: Ki-67 immunostaining in human breast tumors and its relationship to prognosis. Cancer 67:421-428, 1991 20. GoldblumJR, Shannon R, Kaldijian EP, et al: Immunohistochemical assessment of proliferative activity in adrenocortical neoplasms. Mod Pathol 6:663-668, 1993 21. Pelosi G, Zamboni G, Doglioni C, et al: Immunodetection of proliferating cell nuclear antigen assesses the growth fraction and predicts malignancy in endocrine rumors of the pancreas. Am J Surg Pathol 16:1215-1225, 1992 22. Carr K, Heffess C,Jin L, et al: Immunohistochemical analysis of thyroid carcinomas utilizing antibodies to p53 and Ki-67. Appl Immunohistochem 1:201-207, 1993 23. Kitz K, Knosp E, Koos WT, et al: Proliferation of pituitary adenomas: Measurement by Mab Ki-67. Acta Neurochiurg 53:60-64, 1991 (suppl) 24. Loda M, Lipman J, Cukor B, et al: Nodular foci in parathyroid adenomas and hyperplasias. An immunohistochemical study of proliferative activity. HUM PATHOL 25:1050-1056, 1994 25. Thakker RV: The role of molecular genetics in screening for multiple endocrine neoplasia type 1. Endocrinol Clin North Am 23:117-136, 1994 26. Abbona GC, Papotti M, Gasparri G, et al: Proliferative activity in parathyroid tumors as detected by Ki-67 immunostaining. HUM PATHOL 26:135-138, 1995 27. Katoh R, Bray CE, Suzuki K, et al: Growth activity in hyperplastic and neoplastic human thyroid determined by an immunohistochemical staining procedure using monoclonal antibody MIB-1. HUM PATHOL 26:139-146, 1995
HUMAN PATHOLOGY
Volume 26, No. 2 (February 1995)
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