The diagnostic value of Ki-67 and repp86 in distinguishing between benign and malignant mesothelial proliferations.
Abstract: * 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)
Subject: Immunohistochemistry
Monoclonal antibodies
Medical research
Medicine, Experimental
Authors: Taheri, Zohreh Mohammad
Mehrafza, Mehdi
Mohammadi, Forozan
Khoddami, Maliheh
Bahadori, Moslem
Masjedi, Mohammad Reza
Pub Date: 04/01/2008
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
Accession Number: 230151946
Full Text: 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).

Staining Methods

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.

Statistical Analysis

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.

RESULTS

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).

COMMENT

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)

CONCLUSION

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.

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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: mtaheri@nritld.ac.ir).
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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