The diagnostic value of Ki-67 and repp86 in distinguishing between benign and malignant mesothelial proliferations.
* Context.--The differentiation of benign mesothelial
proliferations from malignant mesotheliomas may be difficult, especially
when evaluating small specimens from pleural biopsies.
Objective.--To explore the potential value of 2 proliferative cell markers, Ki-67 and restrictedly expressed proliferation-associated protein 86 kDa (repp86), in distinguishing between malignant mesothelioma (MM) and benign reactive mesothelial hyperplasia (MH).
Design.--Thirty-six cases of MM from 26 men and 10 women with a mean age of 62.9 years (range, 36-80 years) and 22 cases of benign reactive MH from 14 male and 8 female patients with a mean age of 51.5 years (range, 1588 years) were included in this study. The proliferative status of the lesions was assessed by immunohistochemistry using monoclonal antibodies to Ki-S2 (repp86) and Ki-S5 (Ki-67). The labeling indices were quantified.
Results.--The mean labeling indexes for Ki-67 in MM and benign reactive MH were 24.6% (range, 1%-66%) and 6.23% (range, 0%-25%), respectively. The mean labeling indexes for repp86 in MM and benign reactive MH were 26.3% (range, 0%-50%) and 3.26% (range, 0%-21%), respectively. The average proliferative cell count was significantly higher in MM compared with benign reactive MH (P < .001). Furthermore, both markers showed a significant correlation in their expression in MM and benign reactive MH (r = 77.5, P < .001). Sensitivities of 88% and 92% and specificities of 92% and 94% were obtained at a cutoff point of 9% for Ki-67 and repp86, respectively.
Conclusions.--Used in combination, Ki-67 and repp86 appear to be useful markers in differentiating MM from benign reactive MH.
(Arch Pathol Lab Med. 2008;132:694-697)
Taheri, Zohreh Mohammad
Masjedi, Mohammad Reza
|Publication:||Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2008 College of American Pathologists ISSN: 1543-2165|
|Issue:||Date: April, 2008 Source Volume: 132 Source Issue: 4|
|Product:||Product Code: 8000200 Medical Research; 9105220 Health Research Programs; 8000240 Epilepsy & Muscle Disease R&D NAICS Code: 54171 Research and Development in the Physical, Engineering, and Life Sciences; 92312 Administration of Public Health Programs|
The histologic distinction between reactive and neoplastic
mesothelial proliferations can be problematic. True stromal invasion is
the most reliable criterion of mesothelial malignancy; however, invasion
may be difficult to assess, especially in small biopsy specimens. (1-3)
There is currently no reliable immunohistochemical marker that can
distinguish reactive from neoplastic mesothelial cells. In fact, the
United States-Canadian Mesothelioma Reference Panel could not reach a
consensus regarding the benign or malignant nature of mesothelial
proliferations in 22% of 217 cases reviewed between 1994 and 1999.3
Several ancillary techniques, such as flow cytometry for DNA ploidy, p53
immunohistochemistry (IHC), and assessment of telomerase activity, have
been suggested for the resolution of this diagnostic problem. However,
none of these techniques has shown reproducible results. (4-12)
Proliferation markers have been used to aid in the diagnosis of various benign and malignant lesions and to predict patient survival for a variety of tumors. (13-17) However, only a handful of reports have addressed the possible value of the proliferative activity of mesothelial lesions. In this study, we compared the cell cycle status of malignant mesothelioma (MM) with that of benign reactive mesothelial hyperplasia (MH) using IHC for Ki-67 and restrictedly expressed proliferation-associated protein 86 kDa (repp86). Ki-67 antigen is expressed in all stages of the cell cycle except [G.sub.0]. Repp86 (formerly p100) is a cell cycle-associated protein detectable at the transit point from [G.sub.1] to S phase and absent at the end of mitosis; therefore, repp86 is a marker for actively proliferative cells. (18-23) To our knowledge, this is the first study in which the expression of repp86 has been used in conjunction with Ki-67 to differentiate and diagnose mesothelial proliferations.
MATERIALS AND METHODS
Thirty-six cases of MM and 22 cases of benign reactive MH were selected from the pathology files for the period 1999 through 2004 of a major referral center for respiratory disease, Masih Daneshvari Hospital, Tehran, Iran. Two pathologists (F.M., Z.M.T.) reviewed the original slides, and all cases with adequate material for IHC studies were selected Clinically, the lesions presented as diffuse or localized masses on conventional radiographs and computed tomography scans. The morphologic diagnosis of MM was based on atypical mesothelial proliferation with evidence of invasion into the pleural stroma, fatty tissue, or lung parenchyma. The diagnosis of MM was made based on both clinical and morphologic characteristics. This diagnosis was supported by positive IHC panels for WT1 (M 3561, mouse monoclonal), calretinin (M 7245, mouse monoclonal), CK 5/6 (M 7237, mouse monoclonal), and HBME-1 (M 3505, mouse monoclonal) and by negative reactions for carcinoembryonic antigen (M 7072, mouse monoclonal), MOC31 (M 3525, mouse monoclonal), and Leu-M1 (M 0733, mouse monoclonal). All antibodies were obtained from Dako Corporation, Glostrup, Denmark.
The 22 cases of benign reactive MH were obtained from patients with the following conditions: parapneumonic effusion or empyema (14), pneumothorax (2), systemic lupus erythematosus (2), rheumatoid arthritis (1), squamous cell carcinoma of the lung (2), and chronic obstructive pulmonary disease and congestive heart failure (1).
For IHC, 4-^m-thick serial sections from representative blocks were mounted on aminopropoyltriethoxy-coated slides, dried overnight at 37[degrees]C, and then incubated at 60[degrees]C for 1 hour. Endogenous peroxidase activity was blocked by incubation with 2% (wt/vol) methanol-peroxidase. Antigen retrieval was performed by boiling the slides in Tris-HCl at pH 9 in an autoclave (120[degrees]C) for 10 minutes. After cooling, the slides were incubated with monoclonal antibodies Ki-S2 (undiluted, Kiel University, Germany) and Ki-S5 (dilution 1:20, Dako) for 50 minutes at room temperature. The immunoreaction was performed by the streptavidin-biotin complex method with 3,3'-diaminobenzidine as a chromogen. For each step in each run, a section of tonsil was used as a positive control. For a negative control, the primary antibody was omitted and replaced by nonimmune mouse serum. The slides were then counterstained with Harris hematoxylin, dehydrated, and mounted.
Interpretation of Immunohistochemical Staining
The immunohistochemical stains were evaluated using a conventional light microscope at X40 and X400 magnifications. The pathologists were blinded to the final diagnosis; in larger specimens, the histologic picture was likely to favor one diagnosis over the other. For IHC evaluation, an area with the most proliferative activity was seen at X40. Then, 300 consecutive cells in this area were counted using the X400 magnification. Positively stained cells, regardless of their staining intensity, were counted for each proliferative marker. The labeling index (LI) for each case was expressed as a percentage of immunoreactive cells. The interobserver variability for estimation of LI for each case and for each antibody was less than 5%. In some cases, when the difference between the 2 pathologists was more than 5%, the evaluation was jointly repeated using a 2-headed microscope. A false-negative immunoreaction was assumed when no IHC staining of mitotic figures was observed on the original and repeat stains.
We used SPSS 12.0 for Windows (SPSS, Inc, Chicago, Ill) for statistical analysis. The nonparametric Mann-Whitney U test was used to compare the 2 groups in relation to proliferative indices. The correlation between the LIs for Ki-S5 and Ki-S2 was evaluated using the Wilcoxon test and the Spearman rank correlation test.
This study included 36 cases of MM (25 epithelioid, 10 mixed, and 1 sarcomatoid) from 26 men and 10 women (male-female ratio, 2.6:1) with a mean age of 62.9 years (range, 36-80 years) and 22 cases of benign reactive MH from 14 male and 8 female patients with a mean age of 51.5 years (range, 15-88 years). The mean LI values for Ki-67 in MM and benign reactive MH were 24.6% (range, 1%-66%) and 6.23% (range, 0%-25%), respectively, and the mean LI values for repp86 in MM and MH were 26.3% (range, 0%-50%) and 3.26% (range 0%-21%), respectively (Table; Figures 1 and 2). Although the 2 groups had some overlap in the lower values, there were statistically significant differences between the LIs for MM and MH (P < .001). There was also a correlation between the expression of the 2 markers in MM and MH (P < .001).
No statistically significant differences were found when comparing the LIs of Ki-67 and repp86 in benign reactive MH (P = .42), epithelioid mesothelioma (P = .11), and mixed-type mesothelioma (P = .91). A repp86 LI of more than 21% was seen in 20 of 36 cases of MM but in none of the 22 cases of benign reactive MH. A Ki-67 LI of more than 25% was seen in 14 of 36 cases of MM but in none of the cases of benign reactive MH.
Using a receiver operating characteristic curve and a cutoff point of 9%, the diagnostic sensitivity and specificity were 88% and 92% for Ki-67, and 92% and 94% for repp86, respectively (Figure 3).
Differentiation between benign and malignant mesothelial proliferations using morphology alone may be difficult. This is particularly true when the tissue sample is small, and adjacent tissues are not available for histologic evaluation of invasiveness. Ancillary techniques, such as estimation of DNA ploidy and IHC for the expression of p53, epithelial membrane antigen, and desmin, have all been used with limited success. (6-14)
In our study, we used the IHC of repp86, a member of the microtubule-associated protein family, and Ki-67 to help differentiate between benign and malignant mesothelial lesions.
Using repp86 and Ki-67, we demonstrated a significant difference between the LIs of benign reactive MH and MM. Both markers were similar and did not demonstrate significant differences when using the Spearman correlation test (r = 0.77, P < .001).
Repp86 is distinct from all other immunohistochemical proliferation markers. (24) It identifies an 86-kDa nuclear proliferation-specific protein, which is selectively expressed during the S, [G.sub.2], and M phases of the cell cycle. (23,24) This proliferation marker is thought to be more reliable for appraisal of the malignant potential of neoplastic processes. (23,24) On the other hand, Ki-67 is expressed throughout the entire cell cycle, including the [G.sub.1] phase. Since the duration of the [G.sub.1] phase is highly variable among different cells, including it may constitute an unreliable element in cellular proliferation kinetics. (24) However, in the present study, there was no significant difference in the expression of Ki-67 and repp86 in the benign reactive MH and MM groups.
A number of studies have shown significant differences in the expression of proliferating cell nuclear antigen (25) and nuclear organizing regions in nonneoplastic and neoplastic mesotheliomas. (26) Neither, however, is considered to be a specific proliferation marker. (24) To our knowledge, only 3 previous studies have investigated the role of Ki-67 in differentiating between MM and MH. (27,28) Sington et al (27) demonstrated that minichromosome maintenance protein (MCM) and Ki-67 can differentiate malignant from benign reactive mesothelial proliferations, with MCM2 showing better results. This may be because MCM proteins are present throughout the cell cycle, as opposed to the variably expressed Ki-67. In their study on pleural cytology specimens, Schonherr et al (28) showed 100% specificity for Ki-67 in differentiating MM from MH at a 20% cutoff level. Lastly, Cakir et al (29) evaluated telomerase activity and Ki-67 to differentiate between MH and MM and found a sensitivity and specificity of 74% and 86%, respectively.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Using a cutoff level of 9% for the proliferating index in regions with the highest proliferative activity, we differentiated MM from MH with a sensitivity and specificity of 88% and 92% for Ki-67 and 92% and 94% for repp86, respectively.
No significant differences were seen between histologic subtypes of MM (mixed and epithelial) in either Ki-67 or repp86 expression. These results are in concordance with the results of Sington et al. (27)
Our study shows a significant correlation between the LIs of tissues stained with Ki-67 and repp86. Immunostaining of these 2 antibodies appears to be a useful adjunct in distinguishing between MM and benign reactive MH.
The authors are grateful to Farzaneh Rahimi, professor of pathology, for providing the repp86 antibody. We also thank Mehdi Kazempoor, MSc, for performing the statistical analysis and Mahbobeh Mesgarha, laboratory technician. This study was supported by the National Research Institute of Tuberculosis and Lung Diseases, Tehran, Iran.
(1.) Battifora H, McCaughey WTE. Tumors of the Serosal Membranes. Washington, DC: Armed Forces Institute of Pathology; 1995:9-14. Atlas of Tumor Pathology, 3rd series, fascicle 15.
(2.) Attanoos RL, Gibbs AR. Pathology of malignant mesothelioma. Histopathology. 1997;30:403-418.
(3.) Churg A, Colby TV, Cagle P, et al, for the US Canadian Mesothelioma Reference Panel. The separation of benign and malignant mesothelial proliferations. Am J Surg Pathol. 2000;24:1183-1200.
(4.) Frierson HF, Mills SE, Legier JF. Flow cytometric analysis of ploidy in immunohistochemically confirmed samples of malignant epithelial mesothelioma. Am J Clin Pathol. 1988;90:240-243.
(5.) Kafiri G, Thomas DM, Shepherd NA, Krause T, Lane DP, Hall PA. P53 expression is common in malignant mesothelioma. Histopathology. 1992;21:331-334.
(6.) Mayall FG, Goddard H, Gibbs AR. P53 immunostaining in the distinction between benign and malignant mesothelial proliferations using formalin-fixed paraffin sections. J Pathol. 1 992;68:377-381.
(7.) Cagle PT, Brown RW, Lebovitz RM. P53 immunostaining in the differentiation of benign processes from malignancy in pleural biopsy specimens. Hum Pathol. 1994;25:443-448.
(8.) Ramael M, Lemmens G, Eardekens C, et al. Immunoreactivity for P53 protein in malignant mesothelioma and non-neoplastic mesothelium. J Pathol. 1992; 168:371-375.
(9.) Metcalf RA, Welsh JA, Bennet WP, et al. P53 and Kirsten-ras mutation in human mesothelioma cell lines. Cancer Res. 1992;52:2610-2615.
(10.) Mor O, Yaron P, Huszar M, et al. Absence of P53 mutation in malignant mesothelioma. Am J Respir Cell Mol Biol. 1997;16:9-13.
(11.) Kumaki F, Kawai T, Churg A, et al. Expression of telomerase reverse transcriptase (TERT) in malignant mesotheliomas. Am J Surg Pathol. 2002;26:365-370.
(12.) Attanoos RL, Griffin A, Gibbs AR. The use of immunohistochemistry in distinguishing reactive from neoplastic mesothelium: a novel use for desmin and comparative evaluation with epithelial membrane antigen, P53, platelet-derived growth factor receptor, P-glycoprotein and Bcl-2. Histopathology. 2003;43:231-238.
(13.) Meyer JS. Growth and cell kinetic measurement in human tumors. Pathol Annu. 1981;16:53-81.
(14.) Wright NA. Cell proliferation in health and disease. In: Antony PP, Macsween RNM, eds. Recent Advances in Histopathology. Vol 12. New York, NY: Churchill Livingstone; 1984:17-33.
(15.) Tubiana M, Courdi A. Cell proliferation kinetics in human solid tumors: relation to probability of metastatic dissemination and long-term survival. Radiother Oncol. 1989;15:1-18.
(16.) Meyer JS, Friedman E, McCrate M, Bauer WC. Prediction of early course of breast carcinoma by thymidine labeling. Cancer. 1983;51:1879-1886.
(17.) Meyer JS, Prioleau PG. S-phase fractions of colorectal carcinomas related to pathologic and clinical features. Cancer. 1981;48:1221-1228.
(18.) Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol. 1984;133:1710-1715.
(19.) Gerdes J, Li L, Schluter C, et al. Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol. 1991;138:867-873.
(20.) Heidebrecht HJ, Buck F, Endl E, et al. Ki-Mcm6, a new monoclonal antibody specific to Mcm6: comparison of the distribution profile of Mcm6 and the Ki-67 antigen. Lab Invest. 2001;81:1163-1165.
(21.) Heidebrecht HJ, Klages SA, Szczepanowski M, et al. Repp86: a human protein associated in the progression of mitosis. Mol Cancer Res. 2003;1:271-279.
(22.) HeidebrechtHJ, Buck F, Steinmann J, Sprenger R, Wacker HH, Parwaresch R. p100: a novel proliferation-associated nuclear protein specifically restricted to cell cycle phases S, G2 and M. Blood. 1997;90:226-233.
(23.) Rudolph P, Knuchel R, Endl E, Heidebrecht HJ, Hofstader F, Parwaresch R. The immunohistochemical marker Ki-S2: tissue distribution and cell cycle kinetics of a novel proliferation-specified antigen. Mod Pathol. 1998;1 1:450-456.
(24.) Bonatz G, Luttges J, Hedderich J, et al. Prognostic significance of a novel proliferation marker, anti-repp 86, for endometrial carcinoma: a multivariate study. Hum Pathol. 1999;30:949-956.
(25.) Ramael M, Jacobs W, Weyler J, et al. Proliferation in malignant mesothelioma as determined by mitosis counts and immunoreactivity for proliferating cell nuclear antigen (PCNA). J Pathol. 1994;72:247-253.
(26.) Wolanski KD, Whitaker D, Shilikin KB, Henderson DW. The use of epithelial membrane antigen and silver-stained nuclear organizer regions testing in the differential diagnosis of mesothelioma from benign reactive mesotheliosis. Cancer. 1998;82:583-590.
(27.) Sington JD, Morris LS, Nicholson AG, Coleman N. Assessment of cell cycle state may facilitate the histopathological diagnosis of malignant mesothelioma. Histopathology. 2003;42:498-502.
(28.) Schonherr A, Bayer M, Blocking A. Diagnostic and prognostic value of Ki-67 proliferation fraction in serous effusions. Cell Oncol. 2004;26:57-62.
(29.) Cakir C, Gulluoglu MG, Yilmazbayhan D. Cell proliferation rate and telomerase activity in the differential diagnosis between benign and malignant mesothelial proliferations. Pathology. 2006;38:10-15.
Zohreh Mohammad Taheri, MD; Mehdi Mehrafza, MD; Forozan Mohammadi, MD; Maliheh Khoddami, MD; Moslem Bahadori, MD; Mohammad Reza Masjedi, MD
Accepted for publication October 25, 2007.
From the Departments of Pathology (Drs Mohammad Taheri, Mehrafza, Mohammadi, Khoddami, and Bahadori) and Pulmonology (Dr Masjedi), National Research Institute of Tuberculosis and Lung Disease, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: Zohreh Mohammad Taheri, MD, National Research Institute of Tuberculosis and Lung Disease, Department of Pathology, Maseeh Daneshvari Hospital, Darabad Ave, Tehran, Iran (e-mail: email@example.com).
Mean Labeling Index and Range of Expression for Restrictedly Expressed Proliferation-Associated Protein 86 kDa (repp86) and Ki-67 in Benign Reactive Mesothelial Hyperplasia (MH) and Malignant Mesothelioma repp86 Ki-67 Mean, Range, Mean, Range, Diagnosis % % % % Benign reactive MH 3.26 0-21 6.23 0-25 Malignant mesothelioma 26.3 0-50 24.6 1-66
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