Journal of the Egyptian National Cancer Institute (2011) 23, 155–162

Cairo University

Journal of the Egyptian National Cancer Institute

www.nci.cu.adu.egwww.sciencedirect.com

ORIGINAL ARTICLE

Diagnostic value of p53 and ki67 immunostaining for

distinguishing benign from malignant serous effusions

Nesreen H. Hafez *, Neveen S. Tahoun

The Department of Pathology, Cytopathology Unit, National Cancer Institute (NCI), Cairo University, Egypt

Received 14 July 2011; accepted 25 October 2011

Available online 15 December 2011

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KEYWORDS

Serous effusions;

Cytology;

p53;

ki67 immunocytochemistry

Corresponding author. Tel.:

mail address: nesreennci@ho

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i:10.1016/j.jnci.2011.11.001

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Abstract Background: The differentiation of benign mesothelial cells from malignant tumor cells,

primary, or metastatic, in serous effusions based on cytomorphologic features alone can be prob-

lematic.

Purpose: This study was conducted to evaluate the utility of p53 and ki67 immunocytochemical

markers in differentiating benign from malignant tumor cells in serous effusions.

Patients and methods: Archival Papanicolaou-stained smears of 91 pleural and peritoneal effusions

were retrieved from Cytology Unit, Pathology Department, NCI, Cairo University between 2008

and 2010. Forty-one cases were positive for malignant cells and 50 cases were benign based on cyto-

morphologic features. Cases having doubt were excluded from the study. The slides were destained

and subjected to immunocytochemical staining for p53 and ki67. Histologic sections of colonic car-

cinoma and tonsillar tissue were used as positive control for p53 and ki67, respectively. Smears hav-

ing >5% positively stained nuclei for p53 were taken as positive and labeling index P10% of ki67

was considered positive. Frequencies of the individual immunocytochemical stains; p53 and ki67, in

benign and malignant effusion as well as the combination of both stains were calculated.

Results: p53 immunostaining showed nuclear positivity in 31 out of 41 malignant effusions (75.6%)

and in 3 out of 50 benign effusions (6%), p< 0.005. p53 had 75.6% sensitivity, 94% specificity,

91.2% PPV, and 82.5% NPV. ki67 immunostaining was positive in 30 out of 41 malignant effusions

(73.2%) and in 17 out of 50 benign effusions (34%), p< 0.05. ki67 had 73.2% sensitivity, 66%

specificity, 63.8% PPV, and 75% NPV. Cases were then analyzed for combined immunoprofile

of p53 and ki67. Among the 24 cases that coexpressed both antigens, 22 cases (91.7%) were

2607376/0104959148.

(N.H. Hafez).

Institute, Cairo University.

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University.

lsevier

156 N.H. Hafez, N.S. Tahoun

malignant. Thirty two out of 34 cases (94.1%) that showed negative results for both antigens were

benign. For the cases that showed p53 immunostaining only, 9 out of 10 cases (90%) were malig-

nant. Fifteen out of 23 cases (65.2%) that showed ki67 immunostaining were benign.

Conclusion: Benign and malignant effusions showed significantly different staining pattern for p53

and ki67. When used individually, p53 immunostaining can truly diagnose 75.6% and 94% of the

malignant and benign cases, respectively. ki67 immunostaining can correctly identify 73.2% and

66% of the malignant and benign cases, respectively. When used in combination, 91.7% of p53

and ki67 positive cases were malignant while 94% of p53 and ki67 negative cases were benign.

Hence they could be used when the cytomorphology fails to provide a definitive diagnosis.

ª 2011 National Cancer Institute, Cairo University. Production and hosting by Elsevier B.V.

Open access under CC BY-NC-ND license.

Introduction

Identification of malignant, metastatic, or primary malignant

mesothelial cells in serous effusions using cytomorphology aloneis a well known diagnostic problem and is challenging to cytop-athologists [1]. Reactive mesothelial cells in response to many

benign conditionsmay be difficult to distinguish frommalignantcells particularlywhen the former cells occur in clumps and showvarious degree of cytologic atypia [2]. The cytologic featurescommonly used to identify malignancy, including nuclear pleo-

morphism, macronucleoli, large cellular aggregates, papillary-like tissue fragment, and cell in cell engulfment are helpful fea-tures but have limited use in effusion because they may also be

present in florid reactive mesothelial hyperplasia [3].Thus, the availability of techniques that would enhance the

diagnostic accuracy of routine cytological methods could be of

great clinical value. Ancillary techniques such as electronmicroscope, flow cytometry, and morphometry have been usedto solve the ambiguity in cytological differential diagnosis.

However, these are of limited use owing to the high cost, avail-ability at only few specialized centers, requirement of highlyskilled personnel, and low sensitivity and specificity [4]. Theapplication of immunocytochemical technique to serous effu-

sion is more sensitive and specific than other methods and easyavailability of reagents for use in most of the laboratoriesmake it a good choice [5].

Some studies have suggested that the protein product oftumor suppressor gene p53 has been found to be overexpres-sed more frequently in malignant cells than reactive mesothe-

lial proliferation [6,7]. The proliferation cell markers havebeen also used to aid the diagnosis of various benign andmalignant tumors and to predict patient’s survival for a vari-

ety of tumors. The proliferation marker ki67 may be useful inseparating mesothelial proliferation from malignant cells[8,9].

p53 is a 53-kDa nuclear protein product of tumor suppres-

sor gene p53 that is located on the short arm of chromosome17. It is involved in several cellular functions including tran-scription, regulation of the cell cycle, DNA repair, and induc-

tion of apoptosis to preserve the genetic stability. Deletions ormutations in p53 gene are common in human malignancies(60%) leading to tumor growth [10]. The resultant mutated

p53 gene product has a longer half life than the normal proteinand increased stability of aberrant p53 proteins renders themmore readily detectable by immunostaining means. Thus thehighly increased level of mutated gene product in malignant

cells differentiates them from benign cells [11].

ki67 antigen is a cell proliferation-related non-histonenuclear protein that can be labeled with monoclonal antibody

MIB-1. The antigen is expressed in the nuclei of cells in activephases of cell cycle (G1, S, G2, M) except resting phase (G0).ki67 can be used to assess the growth fraction (the number ofcells in cell cycle) of normal, reactive, and neoplastic tissue

[12]. The percentage of ki67 positive cells (labeling index) isusually low in benign lesions but increases in malignanttumors. High ki67 index is an excellent marker to recognize

rapidly proliferating cell that would indicate malignancy andmight affect recurrence rate and survival [13].

The aim of the current study is to evaluate the utility of p53

and ki67 immunocytochemical markers in differentiatingbenign mesothelial cell proliferation from malignant tumorcells, primary, or metastatic, in serous effusions.

Patients and methods

Archival Papanicolaou-stained smears of 91 pleural and peri-

toneal effusions were retrieved from the Cytology Unit,Pathology Department, National Cancer Institute, CairoUniversity between 2008 and 2010. Forty-one cases were posi-tive for malignant cells and 50 cases contained benign reactive

mesothelial hyperplasia. No cases having doubt were includedin the study. Effusions were labeled as benign or malignant onthe basis of cytological examination using the standard cyto-

morphologic features for cytologic diagnosis of serous effusion[14].

Two cases of malignant effusions had confirmatory histo-

logic diagnosis of malignant mesothelioma. The remainingmalignant effusions were confirmed with the previous and/orthe current clinical and radiological findings reviewed fromthe hospital records that showed:

(a) Presence of primary tumor elsewhere.(b) Wide spread metastasis at the time of taping.

(c) Presence of bloody effusion in the absence of traumatictap.

(d) Presence of hemosidren laden macrophages on cytologic

examination that indicated chronic blood leaks.(e) Rapid reaccumulation of the effusions after taping.(f) Poor general condition (cachexia).

The slides were destained using the technique described byMiller and Kubier [15]. The destained slides were subjected toimmunocytochemical staining for p53 and ki67 according to

the streptavidin–biotin–peroxidase technique using the mousemonoclonal antibody Thermo Scientific Lab Vision, anti-p53

Figure 1 Reactive mesothelial cells in pleural effusion showing

variation in size, dense cytoplasm which tends to fade at the

periphery, smooth outer contour, and central or eccentric nucleus

(Papanicolaou stain 400·).

Role of P53 and ki67 immunocytochemistry in serous effusions 157

Ab-6, clone DO-1 (dilution 1:30) and rabbit monoclonal anti-body Thermo Scientific Lab Vision, anti-ki67, clone SP6 (dilu-tion 1:100).

A positive control slide was run with each staining set toensure that all reagents were working properly. Histologic sec-tions of colonic carcinoma and tonsillar tissue were used as

positive control for p53 and ki67, respectively; and a negativecontrol was used by substituting phosphate buffered saline(PBS) for the primary antibody to evaluate non-specific stain-

ing and better interpretation of specific staining. All controlsyielded appropriate results. Only the nuclear immunoreactivityfor both p53 and ki67 was considered specific. Cytoplasmicand membranous staining was considered non-specific. All

slides were immunocytochemically evaluated without anyinformation of clinical, cytopathological, or radiological data.

The result of p53 immunocytochemical stain was scored as:

negative if there was no nuclear staining in the examinedepithelioid cells, focal positive if there was nuclear staining in65% of cells, and positive if there was nuclear staining in

>5% of cells. For ki67, the percentage of positively stainednuclei out of the total epithelioid cell counted was estimated(the labeling index) and categorized as: negative if no nuclear

staining in the examined epithelioid cells, low if there was nu-clear staining in 610% of cells, moderate if there was nuclearstaining in 10–40% of cells, and high if there was nuclear stain-ing in >40% of cells.

For all immunocytochemical stains, the resultswere indepen-dently scored by the two cytopathologists and any discrepantcases were reviewed at a double-headed microscope to achieve

consensus. Statistical analysis of the individual immunocyto-chemical stains; p53 and ki67, in benign andmalignant effusionsas well as the combination of both stains were calculated using

statistical package for social science (SPSS), version 12.

Figure 2 Metastatic high grade undifferentiated carcinoma cells

in pleural effusion showing pleomorphism, irregular nuclear

membrane, and chromatin clearance (Papanicolaou stain 400·).

Table 1 Clinico-pathological causes of the studied malignant

effusion cases.

Clinico-pathological causes Pleural Peritoneal Total

Malignant mesothelioma 2 0 2 (4.9%)

Breast carcinoma 17 0 17 (41.5%)

Lung carcinoma 11 0 11 (26.8%)

Ovarian carcinoma 0 6 6 (14.6%)

Colonic carcinoma 0 3 3 (7.3%)

Gastric carcinoma 1 1 2 (4.9%)

Total 31 10 41

Results

The 41 cases of malignant effusion consisted of 26 females(63.4%) and 15 males (36.6%) with an age range of 44–76 years (mean age was 53.9 years and median age was

56 years). The 50 cases of benign effusion consisted of 28 males(56%) and 22 females (44%) with an age range 31–62 years(mean age was 45.2 years and median age was 49 years).

Twenty-nine cases of the 50 benign effusions (58%) were

developed in the pleural cavity (Fig. 1), while the remaining21 cases (42%) were developed in peritoneal cavity. The malig-nant effusions had developed in the pleural, 31 cases (75.6%)

(Fig. 2), and peritoneal, 10 cases (24.4%), cavities due to manycauses (Table 1).

For cases of malignant and benign effusions, p53 immunocy-

tochemical staining results are shown in Table 2 and Figs. 3–5.Smears having >5% positively stained nuclei were taken aspositive. p53was significantly more expressed in malignant thanbenign effusions (p < 0.0001).

The p53 immunocytochemical results were then comparedwith the clinico-cytological diagnosis of the correspondingcases. The results are presented in Table 3.

p53 had 75.6% sensitivity, 94% specificity, 91.2% positivepredictive value (PPV), and 82.5% negative predictive value(NPV) (Table 6).

The proliferation marker ki67 immunostaining was used todetermine the labeling index. For the studied malignant and

benign effusions, the ki67 labeling index is shown in Table 4

and Figs. 6–8. When using moderate to high labeling index(P10%) as the cutoff point to be considered as positive, thelabeling index was significantly higher in malignant effusionscompared with benign effusions (p < 0.001).

Table 2 Results of p53 immunocytochemical stain in the 91 studied malignant and benign serous effusions.

Clinico-cytological diagnosis p53 ICC score Total

Negative (0%) Focal (65%) Positive (>5%)

Malignant effusions 2 (4.9%) 8 (19.5%) 31 (75.6%) 41

Benign effusions 41(82%) 6 (12%) 3 (6%) 50

ICC, immunocytochemistry.

Figure 3 Metastatic undifferentiated carcinoma to pleural effu-

sion exhibiting positive nuclear staining for p53 (avidin–biotin–

peroxidase 400·).

Figure 4 Metastatic adenocarcinoma to peritoneal effusions

exhibiting positive nuclear staining to p53 (avidin–biotin–perox-

idase 400·).

Figure 5 Metastatic adenocarcinoma to pleural effusion exhib-

iting negative staining for p53, <5% positive nuclei (avidin–

biotin–peroxidase 400·).

Table 3 Comparative analysis of p53 immunocytochemical

staining results and clinico-cytological diagnosis.

Clinico-cytological diagnosis p53 ICC staining

Positive Negative

Positive 31 (91.2%) TP 10 (17.5%) FN

Negative 3 (8.8%) FP 47 (82.5%) TN

Total 34 (100%) 57 (100%)

ICC, immunocytochemistry; TP, true positive cases; FN, false

negative cases; FP, false positive cases; TN, true negative cases.

158 N.H. Hafez, N.S. Tahoun

The ki67 immunocytochemical results were then comparedwith the clinico-cytological diagnosis of the corresponding

cases. The results are presented in Table 5.ki67 had 73.2% sensitivity, 66% specificity, 63.8% positive

predictive value (PPV), and 75% negative predictive value

(NPV) (Table 6).Cases were analyzed for combined immunoprofile of p53

and ki67 ( Table 7). When this comparative analysis was

correlated with the clinic-cytological diagnosis, the results arepresented in Table 8.

From these results, when the studied cases revealed positive

immunostaining reaction for both markers used, there was91.2% probability for malignancy. On the other hand, whenthe studied cases did not express any immunostaining reactionfor the two markers used, 94.1% probability to be benign was

detected. When the studied cases expressed one marker only,the probability of being benign or malignant was nearly equal(48.5% and 51.5%, respectively).

Discussion

Several studies have suggested that p53 immunostaining does

not occur in benign mesothelium but is common in malignan-cies involving serous effusions [7,16,17].

Table 6 Diagnostic reliability of p53 and ki67 immunocyto-

chemical staining on serous effusions.

Parameters p53 (CI) ki67 (CI)

Sensitivity 75.6% (59.5–87.6) 73.2% (56.9–85.7)

Specificity 94% (83.2–98.7) 66% (51.2–78.8)

PPV 91.2% (76.2–98.1) 63.8% (46.4–80. 3)

NPV 82.5% (70.0–91.2) 75% (62.1–85.9)

PPV, positive predictive value; NPV, negative predictive value; CI,

95% confidence interval.

Table 4 Results of ki67 immunocytochemical stain in the 91 studied malignant and benign serous effusions.

Clinico-cytological diagnosis ki67 ICC labeling index Total

Negative (0%) Low (<10%) Moderate (10–40%) High (>40%)

Malignant effusion 0 11 (26.8%) 22 (53.7%) 8 (19.5%) 41

Benign effusions 21 (42%) 12 (24%) 17 (34%) 0 50

ICC, immunocytochemistry.

Figure 6 Metastatic undifferentiated carcinoma to pleural effu-

sion exhibiting positive nuclear staining for ki67 (avidin–biotin–

peroxidase 400·).

Figure 7 Metastatic undifferentiated carcinoma to pleural effu-

sion exhibiting positive nuclear staining for ki67 (avidin–biotin–

peroxidase 400·).

Figure 8 Metastatic adenocarcinoma to peritoneal effusion

exhibiting negative staining for ki67, <10% labeling index

(avidin–biotin–peroxidase 400·).

Table 5 Comparative analysis of ki67 immunocytochemical

staining results and clinico-cytological diagnosis.

Clinico-cytological diagnosis ki67 ICC staining

Positive Negative

Positive 30 (63.8%) TP 11 (25%) FN

Negative 17 (36.2%) FP 33 (75%) TN

Total 47 (100%) 44 (100%)

ICC, immunocytochemistry; TP, true positive cases; FN, false

negative cases; FP, false positive cases; TN, true negative cases.

Role of P53 and ki67 immunocytochemistry in serous effusions 159

In an attempt to differentiate malignant from benign effu-

sions, p53 immunocytochemical stain had been advocated as amalignant marker in 91 pleural and peritoneal serous effusions.p53 was significantly more expressed in malignant than benigneffusions (p< 0.0001). p53 positivity rate was found in 31 of

41 (75.6%) malignant effusions (true positive) and in only 3of 50 (6%) benign effusions (false positive). Our findings arein close comparison with the experience of Hall et al. [18] who

reported that 71% of their studied cytomorphologically

malignant cases were p53 positive and none of the non-malig-

nant effusions showed p53 positivity. Akhtar et al. [19] reported62% positivity rate in malignant effusions while no positivitywas recorded in benign cases. Our positivity rate was much

higher than that reported byMayall et al. [16] who reported that

Table 7 Comparison between p53 and ki67 immunostaining

results.

ki67 ICC results p53 ICC results Total

Positive Negative

Positive 24 23 47

Negative 10 34 44

Total 34 57 91

ICC, immunocytochemistry.

160 N.H. Hafez, N.S. Tahoun

the positivity rate of p53 was found in 17 of 35 (48.6%) malig-nant effusions but was not found in any of 115 benign effusions.Nearly the same results were concluded byHasteh et al. [20] and

Cagle et al. [21] who showed 46.7% and 48% positivity rate inmalignant effusions, respectively, and 2.2% and 0% positivityrate in benign cases, respectively. The higher positivity rate inthe present study, compared to these studies, may be attributed

to the difference in the antibodies used where clone DO-1MoAb had been used in the current study. VPp958 MoAbwas used by Hasteh et al. [20], clone BP53-12 MoAb was used

by Cagle et al. [21] and Do-7 MoAb was used by Mayall et al.[16]. Also in Hasteh et al. study [20], all malignant cases hadmesothelioma and in Cagle et al. [21], 69% of their studied

malignant cases had mesothelioma, while in the present studyonly two cases had malignant mesothelioma. Mayall et al. [22]reported that malignant mesothelioma had low incidence of

p53 mutation. The difference in positivity rate may be alsoattributed to the fact that there is no generalized accepted stan-dard percentage to define the positivity for p53. The authors ap-plied cutoff level>10%positive nuclei to be positive test. In the

current study smears having >5% positive nuclei were consid-ered positive [16,20].

Stoetzer et al. [23] investigated four different monoclonal

antibodies against p53 for the diagnosis of malignancy in effu-sions. They reported that the used antibodies reacted with 52–75% of malignant effusions but also with 38–80% of benign

effusions. They concluded that the sensitivity and specificityof p53 staining in effusions depend strongly on the antibodyused. They finally concluded that p53 staining did not improve

the identification of malignant cells in serous effusions. Thesame conclusion was reached by Walts et al. [7] who reportedp53 reactivity in 78% of malignant effusion, but they also re-ported that the benign mesothelial cells in 14% of the studied

malignant effusions were also stained positively for p53. Inaddition, p53 was positive in 73% of their studied benign effu-sions. They finally concluded that p53 overexpression was not

necessarily indicative of malignancy.Mullick et al. [24] demonstrated p53 positivity in only 41 of

75 (55%) malignant effusions, in 1 of 9 (11%) benign cases,

and in 3 of 19 suspicious cases. This positive benign case wassubsequently diagnosed as malignant mesothelioma on pleural

Table 8 Relation between clinico-cytological diagnosis an

benign effusions.

Clinico-cytological diagnosis +ve for both �Malignant effusion 22

Benign effusion 2 3

Total 24 3

biopsy, while 2 of the 3 positive suspicious cases showed non-small cell lung carcinoma and poorly differentiated large cellcarcinoma. They concluded that positive staining in benign

and suspicious cells warrants further diagnostic evaluation ofthe patients and negative p53 protein immunostaining doesnot exclude malignancy. In the current work, we failed to

prove malignancy in the 3 (6%) positively stained benign cases.One case lost follow up examination at the institute after tap-ing, while the other two cases showed no malignancy on follow

up after reviewing of their hospital records.Compared to our study, Pindzola et al. [25] investigated the

utility of p53 immunostaining to distinguish reactive mesothe-lial cells from metastatic malignant ovarian carcinoma in ser-

ous effusions. They estimated both the intensity and thepercentage of positive nuclear staining. They concluded thatthe staining intensity should be considered as a critical param-

eter in this separation, moderate and strong staining intensitywere considered truly positive. The percentage of nuclear stain-ing, in their study, was less reliable parameter as 25% of be-

nign cases were positive by this assessment.In the current work, 3 of 50 benign cases (6%) were positive

for p53 (false positive). The percentage of p53 positive cells in

studied benign cases (10–20%) was far lower than that seen inmalignant cases (45–90%). Although p53 aberrant accumula-tion is usually detected in malignant tumors, it is also detectedin benign lesions characterized by hyperproliferation and

hyperplasia. Increased cell proliferation induced wild-typep53 protein synthesis, which could regulate cell proliferation,down regulate bcl-2, and activate apoptotic pathway [19]. King

et al. [26] mentioned that occasional overexpression of wild-type p53 protein might be detected immunocytochemically inbenign lesions as a result of normal physiological DNA repair

in response to hyperproliferation or DNA damaging agentscausing false positive results. Levine et al. [27] attributed thecause of p53 positivity in benign lesions to the type of the

monoclonal antibodies used as some antibodies could detectboth the wild- and mutant-type p53 proteins.

In the current work, there were 10 malignant cases (24.4%)that failed to express p53 positivity (false negative cases). The

cause of false negative results could be related to technical fac-tors that reduced or masked the p53 expression in malignanttumors. These factors include methods of cell preparation

(smear versus cell block), methods of immunostaining (un-stained or destained smears), interpretation subjectivity, andthe sensitivity of MoAb used [20].

In the present study, the sensitivity, specificity, PPV, andNPV of p53 immunocytochemical stain were 75.6%, 94%,91.2%, and 82.5%, respectively. Our results are comparableto those of Kafiri et al. [10] who reported 70% sensitivity,

100% specificity, 100% PPV, and 77% NPV. Some previousstudies had reported moderate sensitivity with high specificityfor p53 markers. Akhtar et al. [19] in their similar effort

reported sensitivity, specificity, PPV, and NPV of 64%,

d expression of p53 and ki67 markers in malignant and

ve for both +ve for p53 +ve for ki67

2 9 8

2 1 15

4 10 23

Role of P53 and ki67 immunocytochemistry in serous effusions 161

100%, 100%, and 72.4%, respectively. Sundblad et al. [28] re-ported 59% sensitivity and 100% specificity. Other previousworks had reported relatively lower sensitivity with high spec-

ificity for p53 stain. Hasteh et al. [20] reported 47% sensitivityand 98% specificity. Nearly similar results were reported byMayall et al. [16] who reported 48.6% sensitivity, 100% spec-

ificity, 100% PPV, and 86.5% NPV.The diagnostic value of ki67 immunostaining in human tu-

mors [29,30] as well as in benign and malignant mesothelial

proliferation [31] had been widely documented and accepted.In the present study, there was significant difference in ki67

expression between malignant and benign effusions (p < 0.001)when using >10% labeling index as cutoff point. This is in

accordance with the experience of Schonherr et al. [32] who re-ported a higher ki67 labeling index in malignant than benigneffusions.

However, our data showed that ki67 is not completely reli-able to differentiate benign from malignant effusions because17 of 50 benign cases (34%) were positive (false positive cases)

and 11 of 41 malignant cases (26.8%) had <10% labeling in-dex (false negative cases). The expression of proliferative cellmarkers including ki67 in benign cases was explained by the

possible autocrine or paracrine growth factors that upregu-lated the ki67 gene expression and hence increased the prolif-erative indices [33]. This explanation was proved by thedemonstration of ki67 overexpression in normal breast and

pancreatic exocrine cells in other studies [34,35]. Hasteh etal. [20] concluded that the cause of high labeling index in theirbenign cases might be due to the presence of lymphocytes in

the effusions which were frequently positive and could causedifficulty in estimation of labeling index by immunostaining.In our study some benign cases contained inflammatory cells,

including lymphocytes, in the background. ki67 interpretationwas estimated among the total epithelioid cells counted only,not inflammatory cells.

On the other hand, Saleh et al. [33] attributed the cause oflow labeling index in some malignant cases to the down-regu-lation of ki67 gene by the effect of some autocrine and para-crine uncertain factors. Sikora et al. [36] also attributed the

cause of the false negative cases to the low proliferative andturnover activity in certain tumors or to technical factors thatcause reduction or masking of ki67 expression. These factors

included using destained slides for immunostaining that leadto loss of some antigenicity by the destaining procedure. Kim-ura et al. [37] concluded that percentages of immunostained

cells above and below, but close to, the cutoff point may ac-count for the error in interpretation. They recommended thatthe assessment of immunocytochemistry should be performedusing computer assistance to avoid wrong interpretations.

Other authors attributed the cause of the false negative casesto the low cellularity of the metastatic epithelial cells in theeffusions [20].

In the present study, the sensitivity, specificity, PPV, andNPV of ki67 immunocytochemical stain were 73.2%, 66%,63.8%, and 75%, respectively. Our results are lower than

those reported by Taheri et al. [38] who demonstrated 88%sensitivity and 94% specificity. Taheri et al. [31] in anotherwork evaluated the diagnostic value of ki67 and repp86 in

differentiation of benign mesothelial proliferation frommalignancy and reported 88% sensitivity and 92% specificityfor ki67. Cakir et al. [9] evaluated telomerase activity andki67 immunostaining to differentiate malignant from meso-

thelial proliferation and reported 74% sensitivity and 86%specificity for ki67. Kimura et al. [37] studied three prolifera-tive markers (MCM7, Topo IIa, and ki67) to differentiate

malignant from mesothelial proliferation and reported64.3% sensitivity and 92.9% specificity for ki67 using 30%labeling index as cutoff point. Schonherr et al. [32] in their

similar study on pleural cytology showed 77.8% sensitivityand 90.9% specificity for ki67 when using a cutoff point of>10%, but when they assumed a cutoff point of 25% as po-

sitive test, specificity became 100% but sensitivity was 25%.The improvement of specificity with lowering of sensitivitywhen increasing the cutoff point was also reported by Hastehet al. [20] who estimated 57% sensitivity and 56% specificity

for ki67 when using >10% as cutoff point. They reported16% sensitivity and 91% specificity after using high labelingindex (>40%) as positive test.

In the present study, when assuming >25% labeling indexas cutoff point for positivity; all benign cases were correctlydiagnosed and only 25 of 41 malignant cases (61%) were true

positive. Thus the newly estimated sensitivity and specificitywere 61% and 100%, respectively. Like others, we found thatincreasing the cutoff level could diagnose all benign cases but

miss more malignant cases resulting in increasing specificityand decreasing sensitivity.

Hasteh et al. [20] concluded that each immunostaining mar-ker has its own sensitivity and specificity when used alone. No

single antibody has been shown to be 100% sensitive and abso-lutely specific for identification of malignant cells in serouseffusions. They advised to use a panel of immunocytochemical

stains to make this distinction in cytological effusions. Consis-tent with the role of p53 in controlling cellular proliferation, itwas hypothesized that there was an association between p53

positivity and the number of ki67 positive cells in differentneoplasms, suggesting that aberrant p53 expression is en-hanced by cellular proliferation [23]. To verify whether the

simultaneous association of p53 and ki67 immunodetectioncould help in differentiating malignant from benign effusionswith a high reliability, the comparative expression of bothmarkers was estimated and correlated with clinico-cytological

diagnosis. In the current work, it was important to recognizethat only 24 of the 91 studied cases (26.4%) coexpressed bothmarkers. It was found that for both p53 and ki67 positive

cases, 91.7% were malignant. For cases that were both p53and ki67 negative, 94% were benign. For cases that were p53positive and ki67 negative, 90% were malignant. For cases that

were p53 negative and ki67 positive, 65.2% were benign. p53was considered as a more reliable malignant marker thanki67. For cases that expressed ki67 only, they should be inter-preted cytomorphologically in correlation with clinical and

radiological findings.

Conclusion

Benign and malignant effusions showed significantly differentstaining pattern for p53 and ki67. When used individually,p53 immunostaining can truly diagnose 75.6% and 94% of

the malignant and benign cases, respectively. ki67 immuno-staining can correctly identify 73.2% and 66% of the malignantand benign cases, respectively. When used in combination,

91.7% of p53 and ki67 positive cases were malignant while

162 N.H. Hafez, N.S. Tahoun

94% of p53 and ki67 negative cases were benign. Hence theycould be used when the cytomorphology fails to provide a defin-itive diagnosis.

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