ORIGINAL ARTICLES
Expression and Significance of MOC-31 and Calretinin in Pleural Fluid of Patients With Lung Cancer Ming Lv M.D.,1 Ji-Hong Leng M.D.,1 Yan-Yong Hao M.D.,1* Yan Sun M.D.,1 Na Cha M.D.,2 and Guang-Ping Wu Ph.D.3
Background: The cytologic assessment of pleural effusions to dis-
tinguish carcinoma cells from reactive mesothelial cells is particularly challenging. The aim of this study was to investigate the diagnostic value of monoclonal antibody (MOC-31) and calretinin in pleural fluid of patients with lung cancer to significantly improve the diagnostic accuracy. Methods: The expressions of MOC-31 and calretinin were detected by means of S-P immunocytochemical technique in pleural effusions of patients with lung cancer (n 5 92) and in patients with benign lung disease (n 5 70). Results: The positive rate of MOC-31 in pleural fluid was 90.2% (83/92) from patients with lung cancer and 2.9% (2/70) from patients with benign lung diseases, showing a significant difference (P < 0.01). Calretinin was expressed 87.1% (61/70) in benign lung diseases and 6.5% (6/92) in lung cancer, also showing a significant difference (P < 0.01). The optimal combination for assay was MOC-31 1 calretinin: Sensitivity and specificity were 100 and 98.6%, respectively. Conclusion: MOC-31 and calretinin are of important clinical value for diagnosing and differentially diagnosing the cancer cells in pleural fluid of patients with lung cancer. Diagn. Cytopathol. 2015;43:527–531. VC 2014 Wiley Periodicals, Inc. Key Words: MOC-31; calretinin; immunocytochemistry; tumor markers; lung neoplasms; pleural fluid
1 Department of Pathology, Jilin Cancer Hospital, Changchun, 130012, China 2 Department of Pathology, Changchun Obstetrics Gynecology Hospital, Changchun, 130042, China 3 Department of Pathology, The First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, 110001, China. *Correspondence to: Yan-Yong Hao, M.D., Department of Pathology, Jilin Cancer Hospital, Changchun 130012, China. E-mail: [email protected] Received 16 August 2013; Accepted 6 October 2014 DOI: 10.1002/dc.23218 Published online 24 October 2014 in Wiley Online Library (wileyonlinelibrary.com).
C 2014 WILEY PERIODICALS, INC. V
Pleural effusions are commonly occurring complications produced by a wide variety of diseases. Approximately 20% of pleural effusions are caused by malignancy and in 10–50% of cancer patients may be the initial presentation.1 One of the common difficulties in effusion cytology is the distinction between cancer cells and reactive mesothelial cells (RMCs). Moreover, metastatic adenocarcinoma cells often are associated with prominent mesothelial hyperplasia, making the diagnosis more difficult. The results of the major series reporting the sensitivity of pleural cytology are vary from 40 to 87%, with a mean of about 60%.2–5 Thus, many additional methods have been evaluated to improve the diagnostic accuracy and avoid invasive diagnostic techniques. MOC-31 is a monoclonal antibody that recognizes an epithelial-associated transmembrane glycoprotein of unknown function frequently expressed in epithelial tumors.6 This antibody may help distinguish RMCs from adenocarcinomas in effusion fluids.7–11 Calretinin is a calcium-binding protein that is normally expressed in central and peripheral nervous system neurons,12 and is a reliable and specific marker for mesothelial cells in cytologic preparations.13–16 The main purpose of the study was to determine the diagnostic capacity of MOC-31 and calretinin as tumour markers in pleural effusions. In particular, we assessed the value of MOC-31 and calretinin expression to distinguish between cancer cells and RMCs in pleural effusion specimens.
Materials and Methods Patients The study was conducted according to the regulations of the institutional review boards at Jilin Cancer Hospital. Diagnostic Cytopathology, Vol. 43, No 7
527
Diagnostic Cytopathology DOI 10.1002/dc
LV ET AL.
A total of 162 pleural samples were collected from the patients at the laboratory of cytopathology of the Jilin Cancer Hospital from July 2011 to July 2012. The samples were obtained from 94 (54.7%) male patients, and 78 (45.3%) female patients. A total of 70 effusions were classified as benign, including parapneumonic (n 5 58) and tuberculosis effusions (n 5 12), and 92 were classified as malignant, including adenocarcinomas (n 5 70), squamous cell carcinomas (n 5 8), and small-cell lung cancer (SCLC) effusions (n 5 14). Of the 70 patients with benign effusions, 38 (54.3%) were men and 32 (45.7%) were women, with a mean age of 58.7 years (range, 33–79 years). Of the 92 patients with malignant effusions, 50 (56.5%) were men and 42 (43.5%) were women, with a mean age of 63.4 years (range, 35–92 years). The effusions were classified as benign or malignant on the basis of the criteria described below: Effusions were considered malignant if malignant cells were detected on cytologic examination or in a biopsy specimen. Only specimens from primary malignancies of the lung or pleura were considered; specimens from any other malignancies were excluded. Tuberculous pleurisy was diagnosed if one of the following criteria was met: Identification of bacilli in pleural fluid or biopsy specimen cultures, the presence of caseous granulomas in pleural biopsy tissue, radiologic, and clinical evidence of tuberculous pleurisy with sputum positive for acid-fast bacilli, or response to antituberculous therapy. Parapneumonic pleurisy was diagnosed when there was an acute febrile illness with purulent sputum, pulmonary infiltrates, and responsiveness to antibiotic treatment, or when identification of the microorganism in the pleural effusion in the absence of any other cause of pleural effusions.
Preparation of Cells From Pleural Effusions All specimens were received as fresh effusions, with a volume range of 20–2,000 mL. The specimens were centrifuged for 30 minutes at 2,000g at 4 C. The resulting pellet was used for the preparation of two cytologic smears (alcohol fixed and papanicolaou stained). The rest of the pellet was used for preparation of the paraffinembedded cell block.
Immunocytochemistry For MOC-31 and calretinin staining, 2–4 lm sections were cut from every cell block, deparaffinized in xylene and rehydrated through graded alcohols. Sections were stained in an automated immunostainer (Ventana ES, Ventana Medical System, Inc., Tucson, Arizona, USA.) according to the manufacturer’s instructions. As primary antibodies we used human pancarcinoma-associated epithelial glycoprotein-2 (clone MOC-31, prediluted; Maixin Bio, Fu Zhou, China) and calretinin (clone CR, predi528
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Table I. The Immunocytochemical Stain Results of MOC-31 in Pleural Fluid of Patients With Benign and Malignant Lung Lesions Expression Group Cancer cells Mesothelial cells
n 92 70
a
83 (90.2) 2 (2.9)
Intensity
2
1
11
111
1111
9 68
2 2
5 0
28 0
48 0
Positive (%)
a
P < 0.01 as compared with benign.
Table II. The Immunocytochemical Stain Results of Calretinin in Pleural Fluid of Patients With Benign and Malignant Lung Lesions Expression
Intensity
Group
n
Positive (%)
2
1
11
111
1111
Cancer cells Mesothelial cells
92 70
6 (6.5) 61 (87.1)a
86 9
5 0
1 4
0 7
0 50
a
P < 0.01 as compared with malignant.
luted; Maixin Bio, Fu Zhou, China). For MOC-31, slides were first treated with the pepsin (DIG-3009; Maixin Bio, Fu Zhou, China) for 15 minutes at room temperature. The signal was detected using streptavidin–peroxidase, with 3,3-diaminobenzidine as the chromogen and hematoxylin as the counterstain. A positive result was defined as the presence of stain in >10% of tumor cells in a malignant effusion or in >10% of mesothelial cells in a reactive effusion. Membranous and/or cytoplasmic reactivity was regarded as MOC-31 positive; both nucleus and cytoplasmic reactivity were regarded as calretinin positive. Positive and negative controls were performed on each staining run. The grading of the immunostaining was performed on a sliding scale of 11 to 41 according to the percentage of reactive cells (negative 5 0–10%, 115 11–25%, 21526–50%, 31551–75%, and 41576– 100%).
Statistical Analysis Statistical analysis was performed using the v2 test or Fisher’s exact test when theoretical effectiveness was insufficient. Diagnostic parameters were determined for immunocytochemistry results and compared with those for cytological results. The utility of each marker was determined by means of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy. The level of statistical significance was set at P < 0.05.
Results Results of the marker assay in malignant group and benign group effusions are shown in Table I and Table II. MOC-31 expression was significantly higher in carcinoma than in the benign disease group, whereas calretinin expression was significantly lower in the carcinoma than benign disease group (P < 0.01). MOC-31 staining was
Diagnostic Cytopathology DOI 10.1002/dc
MOC-31 AND CALRETININ IN PLEURAL FLUID Table III. Numbers of Patients Positive for MOC31 Expression With ICC and Cytology in Pleural Effusion of Patients With Benign, Malignant, AC, SCC, and SCLC
Group Benign Malignant AC SCC SCLC
ICC
Cytology
Total no. Patients
1
2
1
2
70 92 70 8 14
2 83a,b 62 7 14
68 9 8 1 0
0 53 42 3 8
70 39 28 5 6
a
P < 0.01 as compared with benign. P < 0.01 as compared with cytology. ICC, immunocytochemistry; SCC, squamous cell carcinoma; AC, adenocarcinoma; SCLC, small cell lung carcinoma. b
Fig. 1. Carcinoma cells showing immunoreactivity for MOC-31 in pleural fluid of a patient with lung cancer. The cancer cells show an atypical glandular cavity-like distribution with intense cytomembrane staining, whereas the reactive mesothelial cells present display negative staining (streptavidin–peroxidase, 3400). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Table IV. Accuracy of ICC for MOC31, CR, and MOC311CR Compared With Cytology in Diagnosis of Pleural Effusion in Patients With Lung Cancer Tumor markers
MOC31 (695% CI)
CR (695% CI)
MOC311CR (695% CI)
Sensitivity Specificity PPV NPV Accuracy
90.2 (66.1)a 97.1 (63.9) 97.6 (63.2) 88.3 (63.7)a 93.2 (62.0)a
87.1 (67.8)a 100 (60.0)a 93.5 (65.0) 98.6 (62.8) 91.0 (66.8) 98.9 (62.1) 90.5 (65.9)a 100 (60.0)a 90.7 (64.5)a 99.3 (61.2)a
Cytology (695% CI) 57.6 (610.1) 100.0 (60.0) 100.0 (60.0) 50.6 (611.0) 70.5 (67.8)
a P < 0.01 ICC compared with cytology. ICC, immunocytochemistry; PPV, positive predictive value; NPV, negative predictive value; CI, confidence intervals.
Fig. 2. Reactive mesothelial cells showing immunoreactivity for calretinin in pleural fluid. The cells show positive nucleus and cytoplasmic staining (streptavidin–peroxidase, 3 400). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
predominantly membranous and/or cytoplasmic staining in cancer cells and with no background staining (Fig. 1). Two cases of benign diseases (2.9%) reacted positively with MOC-31. Calretinin showed strong cytoplasmic and nucleus staining in mesothelial cells (Fig. 2). The results of MOC-31 expression and cytological assessment of pleural fluid specimens from patients with benign or malignant lung disease, including squamous cell carcinoma, adenocarcinoma, and small cell lung carcinoma are presented in Table III. MOC-31 was significantly more likely to be expressed in malignant than in benign lung disease (P < 0.01), and MOC-31 expression was more likely to be positive than cytology in all carcinomas, adenocarcinomas (P < 0.01), and small cell lung
carcinoma (P < 0.05), but not in squamous cell carcinomas (P > 0.05). Table IV depicts the sensitivity, specificity, PPV, NPV, and accuracy of each marker, combinations of markers and cytology examination in pleural fluid. We used MOC-31 for detecting carcinoma cells in the malignant pleural effusions and calretinin for detecting the mesothelial cells in benign pleural effusions, respectively for evaluating the efficiency of each diagnosis in malignant and benign effusions. In single use, MOC-31 had the highest sensitivity (90.2%) and accuracy (97.1%), whereas cytology had the highest specificity (100%) and PPV (100%). When combinations of markers were evaluated together, they gave the highest diagnostic performance: Sensitivity of 100%, NPV (100%), and accuracy of 99.3%, respectively.
Discussion Morphologic differentiation of RMCs from carcinoma cells in pleural effusions can be a diagnostic challenge. In particular, carcinoma metastatic to the pleural membrane is often associated with prominent mesothelial hyperplasia and often results in diagnostic confusion. The difficulty is obviously greater when neoplastic cells show only slight atypia or when they are scarce in the effusion. False negative results of the cytologic examination of pleural fluid Diagnostic Cytopathology, Vol. 43, No 7
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LV ET AL. Table V. Summary of studies of MOC-31 in effusions
Lyons-Boudreaux et al. Su et al.16 Sun et al.23 Kim et al.14 Morgan et al.8 Hyun et al.15 Gong et al.24 Current study Total
13
Carcinoma
Reactive
2/2 (100.0%) 22/32 (68.8%) 54/72 (75.0%) 40/40 (100.0%) 15/17 (88.2%) 11/11 (100.0%) 0/1 (0.0%) 83/92 (90.2%) 227/267 (85.0%)
0/18 (0.0%) 3/35 (8.6) 0/76 (0.0%) 0/30 (0.0%) 0/18 (0.0%) 0/13 (0.0%) 0/2 (0.0%) 2/70 (2.9%) 5/262 (1.9%)
are a serious problem. Such errors in diagnosis usually are caused by misinterpretation of adenocarcinoma cells as RMCs cells.17 The rate of false positive diagnoses is also significant and often caused by overinterpretation of RMCs as malignant cells.7 Immunocytochemistry can greatly aid in such diagnostic dilemmas, but, currently, available markers have varying sensitivities and specificities for mesothelial cells or cells of epithelial differentiation.18,19 MOC-31 is a monoclonal antibody initially raised against a small cell carcinoma cell line. The antibody recognizes an epithelial cell adhesion molecule (Ep-CAM), also known as EGP-2.20 Recent studies have demonstrated MOC-31 reactivity with adenocarcinomas from a variety of primary sites,21 and several studies have described its ability to distinguish adenocarcinomas from RMCs in effusion specimens.7,8,13,22 Kim et al.14 examined serous effusion cell blocks using MOC-31 and found reactivity in 40 of 40 adenocarcinomas (100%) and none of 30 benign effusions. The authors suggest that MOC-31 expression in effusion specimens were useful in the differentiation of adenocarcinomas and RMCs. Likewise, other studies have shown similar results.8,13,15 Sun et al.23 examined MOC-31 reactivity in serous effusion cell blocks and reported reactivity in 54 of 72 adenocarcinomas (75.0%), none of 76 benign effusions (0%), Su et al.16 reported MOC-31 reactivity in 22 of 32 adenocarcinomas (68.8%), 3 of 35 benign effusions (8.6%); also shown resemble results. Otherwise, Gong et al.24 demonstrated the sensitivity was 0%, but only one lung carcinoma in pleural fluid have been detected in their study. Our results are generally comparable to these previous studies (Table V). We observed reactivity for MOC-31 in 83 of 92 carcinoma (90.2%), 2 of 70 reactive effusions (97.1%), with a PPV of 97.6%, and a NPV of 88.3%, especially the number of cases in our study is the largest compared with previous studies. Our study showed that MOC-31 was a useful marker in diagnosing plural effusions with carcinoma. Calretinin is a 29-kd calcium-binding protein that is expressed normally in neurons of the central and periph530
Diagnostic Cytopathology, Vol. 43, No 7
eral nervous system.25 Its role in regulating the cell cycle and cell proliferation is discussed.26,27 Abundant expression of calretinin was first documented in central and peripheral nerval tissue.28 The detection of constant and rather selective expression of calretinin in malignant mesothelioma of the epithelial type,29 respectively in malignant mesothelioma of the epithelial, sarcomatoid, and biphasic types,28 raised the presumption that calretinin is a specific marker of malignant mesothelioma and may also detect benign mesothelial cells.30 The staining pattern of cells of mesothelial origin was cytoplasmic with peripheral condensation and accompanying nuclear staining (Fig. 1). Similar to the observations made by other investigators,28,30–32 the staining of adenocarcinoma cells was focal and less intense when compared with that of reactive and neoplastic mesothelial cells. These reports led us to evaluate the reliability of anticalretinin antibody in benign and metastatic malignant effusions in combination with the epithelial marker MOC-31. Identification of molecular markers for disease progression would be of great clinical value. Immunocytochemistry, the most important ancillary technique used in routine diagnosis of pleural effusions. In the current study, the frequency of MOC-31 expression was significantly greater in the malignant lung disease, squamous cell carcinoma, adenocarcinoma, and small cell lung carcinoma groups (P < 0.01), compared with the benign lung disease group. The rate of positive diagnosis was significantly greater by immunocytochemistry than by cytology in the malignant lung disease, adenocarcinoma groups (P < 0.01), and in the small cell lung carcinoma group (P < 0.05). In the current study, the positive rate of MOC-31 was the highest in small cell lung carcinoma (100%), but definitive conclusions could not be made as there were fewer cases in the group. In conclusion, no single diagnostic technique alone can establish the diagnosis in all cases of body cavity fluid, either malignant or benign. Conventional cytology still has a important role in the diagnosis of serous effusions. For the difficult cases, in which immunocytochemistry is necessary, we believe that the combination of MOC-31 and calretinin can significantly improve the diagnostic accuracy.
References 1. Monte SA, Ehya H, Lang WR. Positive effusion cytology as the initial presentation of malignancy. Acta Cytol 1987;31:448–452. 2. Hirsch A. Pleural effusion: Laboratory tests in 300 cases. Thorax 1979;34:106–112. 3. Salyer WR, Eggleston JC, Erozan YS. Efficacy of pleural needle biopsy and pleural fluid cytopathology in the diagnosis of malignant neoplasm involving the pleura. Chest 1975;67:536–539. 4. Nance KV, Shermer RW, Askin FB. Diagnostic efficacy of pleural biopsy as compared with that of pleural fluid examination. Mod Pathol 1991;4:320–324.
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MOC-31 AND CALRETININ IN PLEURAL FLUID 5. Prakash UB, Reiman HM. Comparison of needle biopsy with cytologic analysis for the evaluation of pleural effusion: Analysis of 414 cases. Mayo Clin Proc 1985;60:158–164. 6. Willuda J, Honegger A, Waibel R, et al. High thermal stability is essential for tumor targeting of antibody fragments: Engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment. Cancer Res 1999;59:5758– 5767. 7. Politi E, Kandaraki C, Apostolopoulou C, Kyritsi T, Koutselini H. Immunocytochemical panel for distinguishing between carcinoma and reactive mesothelial cells in body cavity fluids. Diagn Cytopathol 2005;32:151–155. 8. Morgan RL, De Young BR, McGaughy VR, Niemann TH. MOC31 aids in the differentiation between adenocarcinoma and reactive mesothelial cells. Cancer 1999;87:390–394. 9. Ruitenbeek T, Gouw ASH, Poppema S. Immunocytology of body cavity fluids. MOC-31, a monoclonal antibody discriminating between mesothelial and epithelial cells. Arch Pathol Lab Med 1994;118:265–269. 10. Kundu UR, Krishnamurthy S. Use of the monoclonal antibody MOC-31 as an immunomarker for detecting metastatic adenocarcinoma in effusion cytology. Cancer Cytopathol 2011;119:272–278. 11. Pu RT, Pang Y, Michael CW. Utility of WT-1, p63, MOC31, mesothelin, and cytokeratin (K903 and CK5/6) immunostains in differentiating adenocarcinoma, squamous cell carcinoma, and malignant mesothelioma in effusions. Diagn Cytopathol 2007;36:20–25. 12. Rogers JH. Calretinin. A gene for a novel calcium-binding protein expressed principally in neurons. J Cell Biol 1987;105:1343–1353. 13. Lyons-Boudreaux V, Mody DR, Zhai J, Coffey D. Cytologic malignancy versus benignancy: How useful are the “newer” markers in body fluid cytology? Arch Pathol Lab Med 2008;132:23–28. 14. Kim JH, Kim GE, Choi YD, et al. Immunocytochemical panel for distinguishing between adenocarcinomas and reactive mesothelial cells in effusion cell blocks. Diagn Cytopathol 2009;37:258–261. 15. Hyun TS, Barnes M, Tabatabai ZL. The diagnostic utility of D240, calretinin, CK5/6, desmin and MOC-31 in the differentiation of mesothelioma from adenocarcinoma in pleural effusion cytology. Acta Cytol 2012;56:527–532. 16. Su XY, Li GD, Liu WP, Xie B, Jiang YH. Cytological differential diagnosis among adenocarcinoma, epithelial mesothelioma, and reactive mesothelial cells in serous effusions by immunocytochemistry. Diagn Cytopathol 2010;39:900–908. 17. Afify AM, Stern R, Michael CW. Differentiation of mesothelioma from adenocarcinoma in serous effusions: The role of hyaluronic acid and CD44 localization. Diagn Cytopathol 2005;32:145–150. 18. Ordonez NC. The immunohistochemical diagnosis of mesothelioma: A comparative study of epithelioid mesothelioma and lung adenocarcinoma. Am J Surg Pathol 2003;27:1031–1051.
19. Yaziji H, Battifora H, Barry TS, et al. Evaluation of 12 antibodies for distinguishing epithelioid mesothelioma from adenocarcinoma: Identification of a three-antibody immunohistochemical panel with maximal sensitivity and specificity. Mod Pathol 2006; 19:514–523. 20. Oates J, Edwards C. HBME-1, MOC-31, WT1 and calretinin: An assessment of recently described markers for mesothelioma and adenocarcinoma. Histopathology 2000;36:341–347. 21. Went PT, Lugli A, Meier S, et al. Frequent EpCam protein expression in human carcinomas. Hum Pathol 2004;35:122–128. 22. Hecht JL, Pinkus JL, Pinkus GS. Monoclonal antibody MOC-31 reactivity as a marker for adenocarcinoma in cytologic preparations. Cancer 2006;108:56–59. 23. Sun Y, Wu GP, Fang CQ, Liu SL. Diagnostic utility of MOC-31, HBME-1 and MOC-31mRNA in distinguishing between carcinoma cells and reactive mesothelial cells in pleural effusions. Acta Cytol 2009;53:619–624. 24. Gong Y, Joseph T, Sneige N. Validation of Commonly Used Immunostains on Cell-Transferred Cytologic Specimens. Cancer 2005;105:158–164. 25. Rogers JH. Calretinin: A gene for a novel calcium-binding protein expressed principally in neurons. J Cell Biol 1987;105:1343–1353. 26. Cargnello R, Celio MR, Schwaller B, Gotzos V. Change of calretinin expression in the human colon adenocarcinoma cell line HT29 after dedifferentiation. Biochim Biophys Acta 1996;1313:201–208. 27. Gotzos V, Schwaller B, Hetzel N, Bustos CM, Celio MR. Expression of the calcium binding protein calretinin in WiDr cells and its correlation to their cell cycle. Exp Cell Res 1992;202:292–302. 28. Doglioni C, Laurino L, Chiarelli C, et al. Calretinin: A novel immunocytochemical marker for mesothelioma. Am J Surg Pathol 1996;20:1037–1046. 29. Gotzos V, Vogt P, Celio MR. The calcium binding protein calretinin is a selective marker for malignant pleural mesotheliomas of the epithelial type. Pathol Res Pract 1996;192:137–147. 30. Barberis MC, Faleri M, Veronese S, Casadio C, Viale G. Calretinin. A selective marker of normal and neoplastic mesothelial cells in serous effusions. Acta Cytol 1997;41:1757–1761. 31. Leers MP, Aarts MM, Theunissen PH. E-cadherin and calretinin: A useful combination of immunochemical markers for differentiation between mesothelioma and metastatic adenocarcinoma. Histopathology 1998;32:209–216. 32. Chhieng DC, Yee H, Schaefer D, et al. Calretinin staining pattern aids in the differentiation of mesothelioma from adenocarcinoma in serous effusions. Cancer 2000;90:194–200.
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Expression and Significance of MOC-31 and Calretinin in Pleural Fluid of Patients With Lung Cancer Ming Lv M.D.,1 Ji-Hong Leng M.D.,1 Yan-Yong Hao M.D.,1* Yan Sun M.D.,1 Na Cha M.D.,2 and Guang-Ping Wu Ph.D.3
Background: The cytologic assessment of pleural effusions to dis-
tinguish carcinoma cells from reactive mesothelial cells is particularly challenging. The aim of this study was to investigate the diagnostic value of monoclonal antibody (MOC-31) and calretinin in pleural fluid of patients with lung cancer to significantly improve the diagnostic accuracy. Methods: The expressions of MOC-31 and calretinin were detected by means of S-P immunocytochemical technique in pleural effusions of patients with lung cancer (n 5 92) and in patients with benign lung disease (n 5 70). Results: The positive rate of MOC-31 in pleural fluid was 90.2% (83/92) from patients with lung cancer and 2.9% (2/70) from patients with benign lung diseases, showing a significant difference (P < 0.01). Calretinin was expressed 87.1% (61/70) in benign lung diseases and 6.5% (6/92) in lung cancer, also showing a significant difference (P < 0.01). The optimal combination for assay was MOC-31 1 calretinin: Sensitivity and specificity were 100 and 98.6%, respectively. Conclusion: MOC-31 and calretinin are of important clinical value for diagnosing and differentially diagnosing the cancer cells in pleural fluid of patients with lung cancer. Diagn. Cytopathol. 2015;43:527–531. VC 2014 Wiley Periodicals, Inc. Key Words: MOC-31; calretinin; immunocytochemistry; tumor markers; lung neoplasms; pleural fluid
1 Department of Pathology, Jilin Cancer Hospital, Changchun, 130012, China 2 Department of Pathology, Changchun Obstetrics Gynecology Hospital, Changchun, 130042, China 3 Department of Pathology, The First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, 110001, China. *Correspondence to: Yan-Yong Hao, M.D., Department of Pathology, Jilin Cancer Hospital, Changchun 130012, China. E-mail: [email protected] Received 16 August 2013; Accepted 6 October 2014 DOI: 10.1002/dc.23218 Published online 24 October 2014 in Wiley Online Library (wileyonlinelibrary.com).
C 2014 WILEY PERIODICALS, INC. V
Pleural effusions are commonly occurring complications produced by a wide variety of diseases. Approximately 20% of pleural effusions are caused by malignancy and in 10–50% of cancer patients may be the initial presentation.1 One of the common difficulties in effusion cytology is the distinction between cancer cells and reactive mesothelial cells (RMCs). Moreover, metastatic adenocarcinoma cells often are associated with prominent mesothelial hyperplasia, making the diagnosis more difficult. The results of the major series reporting the sensitivity of pleural cytology are vary from 40 to 87%, with a mean of about 60%.2–5 Thus, many additional methods have been evaluated to improve the diagnostic accuracy and avoid invasive diagnostic techniques. MOC-31 is a monoclonal antibody that recognizes an epithelial-associated transmembrane glycoprotein of unknown function frequently expressed in epithelial tumors.6 This antibody may help distinguish RMCs from adenocarcinomas in effusion fluids.7–11 Calretinin is a calcium-binding protein that is normally expressed in central and peripheral nervous system neurons,12 and is a reliable and specific marker for mesothelial cells in cytologic preparations.13–16 The main purpose of the study was to determine the diagnostic capacity of MOC-31 and calretinin as tumour markers in pleural effusions. In particular, we assessed the value of MOC-31 and calretinin expression to distinguish between cancer cells and RMCs in pleural effusion specimens.
Materials and Methods Patients The study was conducted according to the regulations of the institutional review boards at Jilin Cancer Hospital. Diagnostic Cytopathology, Vol. 43, No 7
527
Diagnostic Cytopathology DOI 10.1002/dc
LV ET AL.
A total of 162 pleural samples were collected from the patients at the laboratory of cytopathology of the Jilin Cancer Hospital from July 2011 to July 2012. The samples were obtained from 94 (54.7%) male patients, and 78 (45.3%) female patients. A total of 70 effusions were classified as benign, including parapneumonic (n 5 58) and tuberculosis effusions (n 5 12), and 92 were classified as malignant, including adenocarcinomas (n 5 70), squamous cell carcinomas (n 5 8), and small-cell lung cancer (SCLC) effusions (n 5 14). Of the 70 patients with benign effusions, 38 (54.3%) were men and 32 (45.7%) were women, with a mean age of 58.7 years (range, 33–79 years). Of the 92 patients with malignant effusions, 50 (56.5%) were men and 42 (43.5%) were women, with a mean age of 63.4 years (range, 35–92 years). The effusions were classified as benign or malignant on the basis of the criteria described below: Effusions were considered malignant if malignant cells were detected on cytologic examination or in a biopsy specimen. Only specimens from primary malignancies of the lung or pleura were considered; specimens from any other malignancies were excluded. Tuberculous pleurisy was diagnosed if one of the following criteria was met: Identification of bacilli in pleural fluid or biopsy specimen cultures, the presence of caseous granulomas in pleural biopsy tissue, radiologic, and clinical evidence of tuberculous pleurisy with sputum positive for acid-fast bacilli, or response to antituberculous therapy. Parapneumonic pleurisy was diagnosed when there was an acute febrile illness with purulent sputum, pulmonary infiltrates, and responsiveness to antibiotic treatment, or when identification of the microorganism in the pleural effusion in the absence of any other cause of pleural effusions.
Preparation of Cells From Pleural Effusions All specimens were received as fresh effusions, with a volume range of 20–2,000 mL. The specimens were centrifuged for 30 minutes at 2,000g at 4 C. The resulting pellet was used for the preparation of two cytologic smears (alcohol fixed and papanicolaou stained). The rest of the pellet was used for preparation of the paraffinembedded cell block.
Immunocytochemistry For MOC-31 and calretinin staining, 2–4 lm sections were cut from every cell block, deparaffinized in xylene and rehydrated through graded alcohols. Sections were stained in an automated immunostainer (Ventana ES, Ventana Medical System, Inc., Tucson, Arizona, USA.) according to the manufacturer’s instructions. As primary antibodies we used human pancarcinoma-associated epithelial glycoprotein-2 (clone MOC-31, prediluted; Maixin Bio, Fu Zhou, China) and calretinin (clone CR, predi528
Diagnostic Cytopathology, Vol. 43, No 7
Table I. The Immunocytochemical Stain Results of MOC-31 in Pleural Fluid of Patients With Benign and Malignant Lung Lesions Expression Group Cancer cells Mesothelial cells
n 92 70
a
83 (90.2) 2 (2.9)
Intensity
2
1
11
111
1111
9 68
2 2
5 0
28 0
48 0
Positive (%)
a
P < 0.01 as compared with benign.
Table II. The Immunocytochemical Stain Results of Calretinin in Pleural Fluid of Patients With Benign and Malignant Lung Lesions Expression
Intensity
Group
n
Positive (%)
2
1
11
111
1111
Cancer cells Mesothelial cells
92 70
6 (6.5) 61 (87.1)a
86 9
5 0
1 4
0 7
0 50
a
P < 0.01 as compared with malignant.
luted; Maixin Bio, Fu Zhou, China). For MOC-31, slides were first treated with the pepsin (DIG-3009; Maixin Bio, Fu Zhou, China) for 15 minutes at room temperature. The signal was detected using streptavidin–peroxidase, with 3,3-diaminobenzidine as the chromogen and hematoxylin as the counterstain. A positive result was defined as the presence of stain in >10% of tumor cells in a malignant effusion or in >10% of mesothelial cells in a reactive effusion. Membranous and/or cytoplasmic reactivity was regarded as MOC-31 positive; both nucleus and cytoplasmic reactivity were regarded as calretinin positive. Positive and negative controls were performed on each staining run. The grading of the immunostaining was performed on a sliding scale of 11 to 41 according to the percentage of reactive cells (negative 5 0–10%, 115 11–25%, 21526–50%, 31551–75%, and 41576– 100%).
Statistical Analysis Statistical analysis was performed using the v2 test or Fisher’s exact test when theoretical effectiveness was insufficient. Diagnostic parameters were determined for immunocytochemistry results and compared with those for cytological results. The utility of each marker was determined by means of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy. The level of statistical significance was set at P < 0.05.
Results Results of the marker assay in malignant group and benign group effusions are shown in Table I and Table II. MOC-31 expression was significantly higher in carcinoma than in the benign disease group, whereas calretinin expression was significantly lower in the carcinoma than benign disease group (P < 0.01). MOC-31 staining was
Diagnostic Cytopathology DOI 10.1002/dc
MOC-31 AND CALRETININ IN PLEURAL FLUID Table III. Numbers of Patients Positive for MOC31 Expression With ICC and Cytology in Pleural Effusion of Patients With Benign, Malignant, AC, SCC, and SCLC
Group Benign Malignant AC SCC SCLC
ICC
Cytology
Total no. Patients
1
2
1
2
70 92 70 8 14
2 83a,b 62 7 14
68 9 8 1 0
0 53 42 3 8
70 39 28 5 6
a
P < 0.01 as compared with benign. P < 0.01 as compared with cytology. ICC, immunocytochemistry; SCC, squamous cell carcinoma; AC, adenocarcinoma; SCLC, small cell lung carcinoma. b
Fig. 1. Carcinoma cells showing immunoreactivity for MOC-31 in pleural fluid of a patient with lung cancer. The cancer cells show an atypical glandular cavity-like distribution with intense cytomembrane staining, whereas the reactive mesothelial cells present display negative staining (streptavidin–peroxidase, 3400). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Table IV. Accuracy of ICC for MOC31, CR, and MOC311CR Compared With Cytology in Diagnosis of Pleural Effusion in Patients With Lung Cancer Tumor markers
MOC31 (695% CI)
CR (695% CI)
MOC311CR (695% CI)
Sensitivity Specificity PPV NPV Accuracy
90.2 (66.1)a 97.1 (63.9) 97.6 (63.2) 88.3 (63.7)a 93.2 (62.0)a
87.1 (67.8)a 100 (60.0)a 93.5 (65.0) 98.6 (62.8) 91.0 (66.8) 98.9 (62.1) 90.5 (65.9)a 100 (60.0)a 90.7 (64.5)a 99.3 (61.2)a
Cytology (695% CI) 57.6 (610.1) 100.0 (60.0) 100.0 (60.0) 50.6 (611.0) 70.5 (67.8)
a P < 0.01 ICC compared with cytology. ICC, immunocytochemistry; PPV, positive predictive value; NPV, negative predictive value; CI, confidence intervals.
Fig. 2. Reactive mesothelial cells showing immunoreactivity for calretinin in pleural fluid. The cells show positive nucleus and cytoplasmic staining (streptavidin–peroxidase, 3 400). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
predominantly membranous and/or cytoplasmic staining in cancer cells and with no background staining (Fig. 1). Two cases of benign diseases (2.9%) reacted positively with MOC-31. Calretinin showed strong cytoplasmic and nucleus staining in mesothelial cells (Fig. 2). The results of MOC-31 expression and cytological assessment of pleural fluid specimens from patients with benign or malignant lung disease, including squamous cell carcinoma, adenocarcinoma, and small cell lung carcinoma are presented in Table III. MOC-31 was significantly more likely to be expressed in malignant than in benign lung disease (P < 0.01), and MOC-31 expression was more likely to be positive than cytology in all carcinomas, adenocarcinomas (P < 0.01), and small cell lung
carcinoma (P < 0.05), but not in squamous cell carcinomas (P > 0.05). Table IV depicts the sensitivity, specificity, PPV, NPV, and accuracy of each marker, combinations of markers and cytology examination in pleural fluid. We used MOC-31 for detecting carcinoma cells in the malignant pleural effusions and calretinin for detecting the mesothelial cells in benign pleural effusions, respectively for evaluating the efficiency of each diagnosis in malignant and benign effusions. In single use, MOC-31 had the highest sensitivity (90.2%) and accuracy (97.1%), whereas cytology had the highest specificity (100%) and PPV (100%). When combinations of markers were evaluated together, they gave the highest diagnostic performance: Sensitivity of 100%, NPV (100%), and accuracy of 99.3%, respectively.
Discussion Morphologic differentiation of RMCs from carcinoma cells in pleural effusions can be a diagnostic challenge. In particular, carcinoma metastatic to the pleural membrane is often associated with prominent mesothelial hyperplasia and often results in diagnostic confusion. The difficulty is obviously greater when neoplastic cells show only slight atypia or when they are scarce in the effusion. False negative results of the cytologic examination of pleural fluid Diagnostic Cytopathology, Vol. 43, No 7
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LV ET AL. Table V. Summary of studies of MOC-31 in effusions
Lyons-Boudreaux et al. Su et al.16 Sun et al.23 Kim et al.14 Morgan et al.8 Hyun et al.15 Gong et al.24 Current study Total
13
Carcinoma
Reactive
2/2 (100.0%) 22/32 (68.8%) 54/72 (75.0%) 40/40 (100.0%) 15/17 (88.2%) 11/11 (100.0%) 0/1 (0.0%) 83/92 (90.2%) 227/267 (85.0%)
0/18 (0.0%) 3/35 (8.6) 0/76 (0.0%) 0/30 (0.0%) 0/18 (0.0%) 0/13 (0.0%) 0/2 (0.0%) 2/70 (2.9%) 5/262 (1.9%)
are a serious problem. Such errors in diagnosis usually are caused by misinterpretation of adenocarcinoma cells as RMCs cells.17 The rate of false positive diagnoses is also significant and often caused by overinterpretation of RMCs as malignant cells.7 Immunocytochemistry can greatly aid in such diagnostic dilemmas, but, currently, available markers have varying sensitivities and specificities for mesothelial cells or cells of epithelial differentiation.18,19 MOC-31 is a monoclonal antibody initially raised against a small cell carcinoma cell line. The antibody recognizes an epithelial cell adhesion molecule (Ep-CAM), also known as EGP-2.20 Recent studies have demonstrated MOC-31 reactivity with adenocarcinomas from a variety of primary sites,21 and several studies have described its ability to distinguish adenocarcinomas from RMCs in effusion specimens.7,8,13,22 Kim et al.14 examined serous effusion cell blocks using MOC-31 and found reactivity in 40 of 40 adenocarcinomas (100%) and none of 30 benign effusions. The authors suggest that MOC-31 expression in effusion specimens were useful in the differentiation of adenocarcinomas and RMCs. Likewise, other studies have shown similar results.8,13,15 Sun et al.23 examined MOC-31 reactivity in serous effusion cell blocks and reported reactivity in 54 of 72 adenocarcinomas (75.0%), none of 76 benign effusions (0%), Su et al.16 reported MOC-31 reactivity in 22 of 32 adenocarcinomas (68.8%), 3 of 35 benign effusions (8.6%); also shown resemble results. Otherwise, Gong et al.24 demonstrated the sensitivity was 0%, but only one lung carcinoma in pleural fluid have been detected in their study. Our results are generally comparable to these previous studies (Table V). We observed reactivity for MOC-31 in 83 of 92 carcinoma (90.2%), 2 of 70 reactive effusions (97.1%), with a PPV of 97.6%, and a NPV of 88.3%, especially the number of cases in our study is the largest compared with previous studies. Our study showed that MOC-31 was a useful marker in diagnosing plural effusions with carcinoma. Calretinin is a 29-kd calcium-binding protein that is expressed normally in neurons of the central and periph530
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eral nervous system.25 Its role in regulating the cell cycle and cell proliferation is discussed.26,27 Abundant expression of calretinin was first documented in central and peripheral nerval tissue.28 The detection of constant and rather selective expression of calretinin in malignant mesothelioma of the epithelial type,29 respectively in malignant mesothelioma of the epithelial, sarcomatoid, and biphasic types,28 raised the presumption that calretinin is a specific marker of malignant mesothelioma and may also detect benign mesothelial cells.30 The staining pattern of cells of mesothelial origin was cytoplasmic with peripheral condensation and accompanying nuclear staining (Fig. 1). Similar to the observations made by other investigators,28,30–32 the staining of adenocarcinoma cells was focal and less intense when compared with that of reactive and neoplastic mesothelial cells. These reports led us to evaluate the reliability of anticalretinin antibody in benign and metastatic malignant effusions in combination with the epithelial marker MOC-31. Identification of molecular markers for disease progression would be of great clinical value. Immunocytochemistry, the most important ancillary technique used in routine diagnosis of pleural effusions. In the current study, the frequency of MOC-31 expression was significantly greater in the malignant lung disease, squamous cell carcinoma, adenocarcinoma, and small cell lung carcinoma groups (P < 0.01), compared with the benign lung disease group. The rate of positive diagnosis was significantly greater by immunocytochemistry than by cytology in the malignant lung disease, adenocarcinoma groups (P < 0.01), and in the small cell lung carcinoma group (P < 0.05). In the current study, the positive rate of MOC-31 was the highest in small cell lung carcinoma (100%), but definitive conclusions could not be made as there were fewer cases in the group. In conclusion, no single diagnostic technique alone can establish the diagnosis in all cases of body cavity fluid, either malignant or benign. Conventional cytology still has a important role in the diagnosis of serous effusions. For the difficult cases, in which immunocytochemistry is necessary, we believe that the combination of MOC-31 and calretinin can significantly improve the diagnostic accuracy.
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