Path. Res. Pract. 186, 658-665 (1990)
The Application of Immunocytochemical Techniques to Routinely-Fixed and Stained Cytologic Specimens An Aid in the Differential Diagnosis of Undifferentiated Malignant Neoplasms J. Weintraub, M. Redard, D. Wenger and P. Vassilakos Center for Cytology and Cancer Screening and Division of Gynecologic and Obstetric Pathology, Department of Pathology, University of Geneva, Switzerland
SUMMARY
Routinely-fixed and Papanicolaou stained smears with the cytologic diagnosis of undifferentiated malignant neoplasm that had been prepared with cells obtained by fine needle aspiration biopsy (n = 7), pulmonary lavage (n = 5), or thoracentesis (n = 3) from 15 unselected patients were stained by an immunocytochemical technique to evaluate the presence of keratin proteins and the leukocyte common antigen (LCA). Commercially available, well-characterized monoclonal antibodies with specificities for keratin proteins and the leukocyte common antigen, and a streptavidin-biotin-horseradish peroxidase labelling method were used. Evaluation of the stained smears revealed the presence ofone of the two antigens in material obtained from each patient, thus indicating the probable cell-lineage ofthe neoplastic cells. The specificity ofthe monoclonal antibody reagents used was further evaluated in routinely-fixed and stained cytologic material from 24 histologically confirmed carcinomas and 12 lymphomas. In conclusion, immunocytochemical techniques may be successfully applied to routinely processed archival cytologic smears to determine the antigenic profile of morphologically undifferentiated cells and therefore aid in the differential diagnosis of undifferentiated malignant neoplasms.
Introduction Immunocytochemical methods using monoclonal antibodies to identify cell-lineage specific antigens have been increasingly applied in routine histopathology as an aid in the differential diagnosis of undifferentiated malignant neoplasms 3,8,22,32. Among the most commonly employed antibodies are those with specificities for intermediate filament proteins present in the cytoplasm of epithelial and mesenchymal cells 20,28 and for the leukocyte common antigen (LeA), a protein present on the surface of hematopoietic cells 18 ,32. The early identification of the cell-lineage of morphologically undifferentiated malignant cells can be an important element in establishing the definitive diagnosis, in providing prognostic information, 0344-0338/90/0186-0658$3.5010
and in contributing to the choice of appropriate therapy7. Immunocytochemical techniques have been widely applied to cytopathology and numerous studies have shown their utility as a diagnostic aid 1,2,5, 19,23,25,27. These studies have generally been performed using fixed and paraffin embedded cells ("cell blocks") or with cytologic smears prepared using special fixation protocols and preparation procedures. Since there is not always sufficient cytological material to prepare cell blocks, nor to anticipate which samples will present difficult diagnostic problems, some authors have investigated the use of routinelyfixed (ethanol 95%) and stained (Papanicolaou) cytologic smears for immunocytochemistry 1,4,25,29. Several studies have shown that monoclonal or polyclonal antibodies may © 1990 by Gustav Fischer Verlag, Sruttgarr
Immunocytochemistry with Routine Cells . 659
be used to detect specific antigens which remain stable after routine processing. These include intermediate filaments 1, 5, 6, 21, epithelial cell and microbial proteins4, 11, 15,21,29, peptide hormones 9, 23, 29,31, leukocyte antigens and immunoglobulins5,23,29, oncofetal proteins I4 ,21,23,29,30, and prostatic tissue antigens 13 ,23. However, only a limited number of reports, based on small numbers of samples, have evaluated the application of antigen detection techniques to the differential diagnosis of morphologically undifferentiated lesions using routinely fixed and stained materia\2 6 • The present investigation was designed to address 2 related problems, one technical and the other diagnostic. Firstly, we have evaluated the reliability of using routinelyfixed and stained cytologic smears to perform immunocytochemical studies using a limited number of well characterized monoclonal antibodies with specificities for keratin proteins and LCA respectively. Secondly, we have applied this technique to determine the presence of cell-lineage specific antigens in a series of 15 morphologically undifferentiated malignant tumors of unknown origin obtained by cytologic sampling methods. The presence of multiple antigens was evaluated simultaneouslyon the same slide.
Material and Methods
Patient Samples Routinely-fixed (ethanol 95% - ether, 1: 1 v/v) and stained (method of Papanicolaou) smears collected during the period 1986-1988 were retrieved from the archives of the Cytology Center and from the Laboratory Clinic, Geneva, Switzerland. The samples had been obtained by fine needle aspiration biopsy (n = 7), pulmonary or peritoneal lavage (n = 5), or thoracentesis (n = 3). Smears were collected from 15 cases with the cytologic diagnosis of undifferentiated malignant neoplasm (Table 1). In all cases a subsequent histopathological examination was performed. Controls for monoclonal antibody specificity included routinely-fixed and stained smears from 24 patients with the cytologic diagnosis of carcinoma (Table 2) and from 12 with the cytological diagnosis of malignant lymphoma (Table 3). All diagnoses were histologically confirmed.
Antibody Reagents and Immunocytochemical Methods The antibodies used in this study, the appropriate dilutions and the respective source of supply are listed in Table 4. For immunocytochemical staining a peroxidase conjugated streptavidin labelling procedure was used12, 19. The details of the staining protocol are shown in Table 5. No specific destaining step was employed to remove the Papanicolaou stain. The cytologic material was subdivided into 3 regions in order to stain simultaneously for several antigens on the same slide. Following removal of the coverslip, a band-like zone, 0.3 cm in width, situated between the different regions, was scraped clean of cytologic material. In this manner, and using a careful technique, different primary antibodies placed on different areas of the slide could be kept from mixing. No additional products, such as mounting medium, were necessary to separate the different regions.
Controls and the Evaluation of Immunostaining A negative control was included with each patient sample studied by omitting the primary antibody (and replacing it with phosphate buffered saline, pH 7.2, containing 5% bovine serum albumin - PBS-5 01o BSA). Readily identifiable benign epithelial and lymphoid elements served as internal positive and negative controls when present in the cytologic preparations. Positive cytoplasmic staining was interpreted as demonstrating the presence of keratin. In contrast, staining of cells for LCA was considered positive when the distribution of labelling was primarily membranous. An estimation of the distribution (proportion of stained neoplastic cells) and intensity of immunostaining (weak, moderate, strong) was performed by classifying at least 200 consecutive neoplastic cells in contiguous fields (when sufficient cytologic material was available). The evaluation of the results of the immunostaining of cases of undifferentiated malignant neoplasm was performed without prior knowledge of the histologic diagnosis. The evaluation of the control series of carcinomas and lymphomas was done by an observer OW) with no knowledge of the cytologic or histopathologic diagnosis. The diagnosis in all cases was histologically confirmed. Cytologic diagnoses were based on standard criteria 17.
Results A total of 15 specimens with the cytologic diagnosis of undifferentiated malignant neoplasm were examined by immunocytochemistry for the presence of keratin proteins and the leukocyte common antigen. In all cases the identification of the product of the staining reaction was unambiguous and in the appropriate cellular localization, i.e. the distribution of staining for keratin was cytoplasmic and for LCA was membranous and cytoplasmic (Fig. 1). Background staining, as a result of non-specific binding was minimal. The results of the immunostaining are presented in Table 1. The antigenic profile of the atypical cells was compatible with an epithelial cell-lineage and the diagnosis of carcinoma in 12 cases and with a leukocyte cell-lineage and the diagnosis of lymphoma in 3 cases. At least 25% of the neoplastic cells were stained in all cases. The staining was moderate to strong. One case (pt 1) showed a punctate pattern of intracytoplasmic staining. In all cases the subsequent histologic diagnosis confirmed the interpretation of the immunocytochemical staining pattern. In this series, the most frequent diagnostic dilemma concerned the distinction between lymphoma and small cell anaplastic carcinoma and, secondly, between large cell carcinoma and immunoblastic lymphoma. In an effort to control the specificity and reliability of the antibody staining reagents, the antigenic profile of neoplastic cells of known origin was determined in a selected series of cases in which the cytologic and histologic diagnoses were available. These included 24 cases of carcinoma and 12 cases of lymphoma. There were 8 small cell carcinomas of the lung, 12 adenocarcinomas from prostate, breast, ovary, stomach and pancreas, 1 hepatocellular carcinoma, 2 squamous cell carcinomas, and 1 transitional cell carcinoma of the bladder (Table 2). The 12 lymphomas included 6 centrocyticlcentroblastic, or well differentiated lymphocytic types, 4 immunoblastic
660 . Weintraub,
J. et a!.
Table 1. Clinico-pathologic features and immunostaining characteristics for 15 cases with the initial cytologic diagnosis of undifferentiated malignant neoplasm Sex/age
Source of cytologic sample
Results of immunostaining Percent positive Definitive histologic diagnosis keratin cells/staining intensity AE l/AE 3 CAM 5.2 LCA
1 2
F/80 Ml73
pulmonary lavage FNABal-iliac fossa
+
+
3 4 5 6
F/65 F/55 F/34 F/64
pulmonary lavage thoracentesis pulmonary lavage pulmonary lavage
+ + + +
+ + + +
50%/++ 100%/+++ 100%/+++ rare cells/+ + +
7
Ml80
FNAB-Iymph node
+
+
100%/++ - +++
8 9
Ml79 Ml61
FNAB-Iung thoracentesis
+ +
+ +
75%/++ 100%/++
10 11
F/64 F/77
peritoneal lavage FNAB-lymph node
+
+
12
Ml74
thoracentesis
13
F/85
+
75%/++
14
Ml74
FNAB-subcutaneous + nodule FNAB-salivary gland +
+
100%/+++
15
Ml69
FNAB-Iymph node
+
100%/+++
Patient number
+
+
75%1++ 25%/+
+
50% 75%/++
+
100%/++
small cell carcinoma of the lung malignant lymphoma-centrocytic type small cell carcinoma of the lung undifferentiated carcinoma pneumoblastoma large cell undifferentiated carcinoma of the lung small cell carcinoma of the bladder small cell carcinoma of the lung poorly differentiated squamous cell carcinoma of the lung endometrial adenocarcinoma malignant lymphoma-B-immunoblastic malignant lymphoma-centroblastic metastatic ductal carcinoma of the breast large cell undifferentiated carcinoma of the lung small cell carcinoma of the lung
a) FNAB: fine needle aspiration biopsy.
Table 2. Clinico-pathologic features and immunostaining characteristics for "known" cases of carcinoma Sex/age
Source of cytologic sample
Keratin % positive cells/staining intensity
Definitive histologic diagnosis
1 2
Ml76 Ml53
pulmonary lavage pulmonary lavage
100%/++ rare neop!. cells/+++
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
F/60 Ml70
pulmonary lavage thoracentesis thoracentesis FNAB-prostate FNAB-prostate FNAB-lung paracentesis FNAB-liver FNAB-Iymph node paracentesis pulmonary lavage pulmonary lavage pulmonary lavage pulmonary lavage thoracentesis thoracentesis peritoneal lavage thoracentesis thoracentesis paracentesis paracentesis thoracentesis
rare neop!. cellsl+ + 50%/++ 50%1++ 25%1+ 25%/+ 50%/++ 100%/++ 75%1++ 50%/++ 90%1++ 75%/+ 50%/+ 50%/+ 50%1+ 100%/+++ 50%1++ 100%/++ - +++ 50%/++ 100%1+++ 100%/++ 100%/++ 100%/+++
small cell carcinoma of the lung small cell and poorly differentiated squamous cell carcinoma of the lung poorly differentiated squamous cell carcinoma of the lung small cell carcinoma of the lung small cell carcinoma of the lung adenocarcinoma of the prostate adenocarcinoma of the prostate small cell carcinoma of the lung papillary transitional carcinoma of the bladder hepatocellular carcinoma infiltrating ductal carcinoma of the breast adenocarcinoma small cell carcinoma of the lung small cell carcinoma of the lung small cell carcinoma of the lung poorly differentiated squamous cell carcinoma infiltrating ductal carcinoma of the breast papillary serous carcinoma of the ovary poorly differentiated ovarian carcinoma infiltrating ductal carcinoma of the breast infiltrating ductal carcinoma of the breast papillary serous cystadenocarcinoma of the ovary moderately differentiated gastric adenocarcinoma infiltrating ductal carcinoma of the breast
Patient number
22
23 24
Min
Ml81 Ml86 Ml79 F/74 Ml76 F/42 F/43 Ml89 Ml75 Ml69 Ml77 F/58 F/64 F/64 F/68 F/73 F/75 Ml60 F/57
Immunocytochemistry with Routine Cells . 661
A
c Fig. 1. Cell smears from 2 lesions with the cytologic diagnosis of undifferentiated malignant neoplasm stained by immunocytochemistry (original magnification x 1070) (Table 1). - A: Patient number 7: neoplastic cells stained for keratin proteins (definitive histologic diagnosis - carcinoma). - B: Patient number 7: neoplastic cells stained for LCA - note positively stained macrophage. - C: Patient number 12: cells stained for keratin; positive cytoplasmic staining is present in 2 mesothelial cells (definitive histologic diagnosis lymphoma). - 0: Patient number 12: Cells stained for LCA; all cells show membrane and some cytoplasmic staining with the exception of 2 mesothelial cells.
662 . Weintraub, J. et al.
Fig. 2. Cell smears from "known" cases of carcinoma and lymphoma stained by immunocytochemistry (original magnification x 1000) (Table 2 and 3). - A: Patient number 8 (Table 2): neoplastic cell stained for keratin proteins - mature lymphocytes are unstained (definitive histologic diagnosis - carcinoma). - B: Patient number 8 (Table 2): neoplastic cells stained for LCA-positive staining is limited to lymphocytes. - C: Patient number 2 (Table 3): neoplastic cells stained for keratin - one mesothelial cell shows cytoplasmic staining (definitive histologic diagnosis -lymphoma). D: Patient number 2 (Table 3): neoplastic cells are uniformly stained for LCA-note absence of staining in one mesothelial cell.
Immunocytochemistry with Routine Cells . 663
types, and 1 case each of multiple myeloma and mixedcellularity Hodgkin's disease (Table 3). The quality of the staining reactions was identical to that observed in the series of morphologically undifferentiated malignant neoplasms. No cases showed simultaneous staining for lymphoid and epithelial antigens and there was no background or nonspecific staining to complicate the evaluation of the immunostaining. All carcinomas stained positively for keratin; there was no staining with LCA. The distribution and intensity of staining varied (Table 1). The lymphomas were uniformly positive for LCA with between 90%-100% of the neoplastic cells stained. The intensity varied from moderate to strong (data not shown). As previously noted, staining for LCA was pronounced along the cell surface, while staining for keratin was cytoplasmic (Fig. 2).
The results obtained in this study confirm and extend previous reports in which the presence of specific antigens in cells present on routinely-fixed and stained smears was evaluated 1,21,29. While the immunostaining protocol used in this investigation was based on a traditional peroxidase labelling procedure, several important technical differences exist with previously reported protocols 29 . These include: 1) the elimination of a specific destaining step which was found to be unnecessary; 2) the use of a streptavidin-biotin-horseradish peroxidase-diamino benzidine labelling system, which virtually eliminated nonspecific background staining; 3) the use of well-characterized and commercially available monoclonal antibody diagnostic reagents to improve specificity of staining; and 4) the ability to stain for several antigens simultaneously
Table 3. Clinico-pathologic features and immunostaining characteristics for "known" cases of malignant lymphoma
1
2 3 4 5
6 7 8 9 10 11
12
1
Sexlage
Source of cytologic Definitive histologic sample diagnosis with subclassification
Ml85 Ml82 Ml63 Ml85 Ml33 F/85 F/83 FI50
paracentesis thoracentesis paracentesis FNABLJymph node FNAB-lymph node paracentesis thoracentesis thoracentesis thoracentesis thoracentesis thoracentesis thoracentesis
Ml49 F/80 F/71
Ml70
Antibody (antigen)
Cellular reactivity
AE lIAE 3 (keratin) CAM 5.2 (keratin)
epithelial tumors epithelial tumors
Dilution sourceI
1: 50 Hybritech 1: 50 Amersham anti-LCA (leukocyte neoplastic leukocytes 1: 20 Dako common antigen, CD 45) 1 : 100 Amerbiotinylated rabbit anti-mouse Ig sham I Hybritech, San Diego CA; Amersham, U.K.; Dako, Glostrup, Denmark.
Table 5. Immunostaining protocol
1. remove coverslip with xylol
Discussion
Patient number
Table 4. Antibodies used in the analysis of cytologic smears
FNAB-fine needle aspiration biopsy.
multiple myeloma well differentiated lymphocytic unclassified centrocytic-centroblastic Hodgkin's disease mixed cellularity centrocytic T-lymphocytic T-Iymphocytic centroblastic centrocytic-centroblastic T-Iymphocytic centrocytic
2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15.
place in 95% ethanol, 2 x 1 minI incubate in methanol- 3% H 20 2, 10 min rehydrate cells in a graded series of ethanols place in PBS2, 5 min incubate in PBS - 5% BSA2, 10 min incubate with specific primary monoclonal antibody, overnight, 4°C place in PBS,S min incubate with biotinylated rabbit anti-mouse Ig2, diluted 1 : 100,30 min place in PBS,S min incubate with streptavidin-biotin-horseradish peroxidase complex 3 diluted 1 : 200, 15 min place in PBS,S min incubate with diaminobenzidine solution4, 5 min rinse with distilled water counterstain and coverslip
I All steps were performed at 20 °c unless otherwise specified. 2 Abbreviations used: PBS, phosphate buffered saline pH 7.2; BSA, bovine serum albumin; Ig, immunoglobulin. - 3 The streptavidin-biotin-horseradish peroxidase complex was obtained from Amersham, U.K. - 4 50 mg diaminobenzidinellOO ml distilled water containing 3% H 20 2.
on the same smear in order to conserve material and to diminish problems resulting from the uneven distribution of neoplastic cells from smear to smear. In addition, little cell loss by detachment from slides was noted during the immunocytochemical staining procedure. Antigenicity, in the case of the antigens evaluated, was maintained for months and, in some cases, years. As a result of these factors, the immunocytochemical signal was clearly evident and easily evaluable; it was uncomplicated by non-specific background staining. This investigation focused on the application of immunocytochemical methods to the differential diagnosis of morphologically undifferentiated malignant neoplasms. Fortuitously, the series of 15 unselected patients studied did not include any in whom the diagnosis of sarcoma or melanoma was subsequently confirmed histopathologically. However, it is clear that these diagnoses and others
664 . Weintraub, J. et al.
must be included in the differential diagnosis of morphologically undifferentiated malignant neoplasms. Further, antibody reagents should be employed which permit the identification of the corresponding cell-lineage. The results of preliminary studies in our laboratory have confirmed the utility of broadening the antibody panel by using reagents specific for the intermediate filament protein, vimentin, and the melanoma associated antigen, HMB45 10,3°. These well-characterized and commercially available monoclonal antibody reagents give a similar quality of labelling as described in this study for antikeratin and anti-LeA antibodies. Once an epithelial cell lineage has been determined, the antibody panel may be broadened still further to determine the organ of origin of metastatic carcinomatous cells. Preliminary investigations have confirmed the utility, in specific clinical situations, of incorporating monoclonal antibody reagents with specificities for prostatic and ovarian antigens 13 ,16. However, our experience with epithelial cell associated antigens has been mixed and the specificity, using routinely fixed and stained material, of some of the antibodies remains to be established 24 • In conclusion, immunocytochemical staining methods may be applied to archival routinely-fixed and stained cell smears in an effort to resolve diagnostic problems.
References 1 Chess Q, Hajdu SI (1986) The role of immunoperoxidase staining in diagnostic cytology. Acta Cytol 30: 1-7 2 Coleman DV, Ormerod MB (1984) Tumor markers in cytology. In: Koss LG (Ed) Advances in Clinical Cytology, Vol 2: 33-47. Year Book Medical Publishers Inc., Chicago 3 Corson JM (1986) Keratin protein immunohistochemistry in surgical pathology practice. In: Sommers SC, Rosen PP, Fechner RE (Eds.) Pathology Annual Part 2: 47-82. Appleton-Century Crofts, Norwalk 4 Dalquen P, Bittel D, Gudet F, Overbeck J, Heitz PU (1986) Combined immunoreaction and Papanicolaou stain on cytologic smears. Path Res Pract 181: 50-54 5 Dinges H-P, Wirnsberger G, Hofler H (1989) Immunocytochemistry in cytology: comparative evaluation of different techniques. Analyt Quant Cytol Histol 11: 22-32 6 Faravelli A, Sironi M, Villa E, Radice F (1984) Immunoperoxidase of cytologic smears in oral pemphigus. Acta Cytol28: 414-418 7 Gatter KC, Alcock C, Heryet A, Mason DY (1985) Clinical importance of analyzing malignant tumours of uncertain origin with immunohistological techniques. Lancet i: 1302-1305 8 Gatter KC, Alcock C, Heryet A, Pulford KA, Heyderman E, Taylor-Papadimitriou J, Stein H, Mason DY (1984) The differential diagnosis of routinely-processed anaplastic tumors using monoclonal antibodies. Am J clin Pathol 82: 33-43 9 Geddie WR, Bedard YC, Strawbridge HTG (1984) Medullary carcinoma of the thyroid in fine-needle aspiration biopsies. Am J Clin Pathol 82: 552-558 10 Gown AM, Vopel AM, Hoak D, Gough F, McNutt MA (1986) Monoclonal antibodies specific for melanocytic tumors distinguish subpopulations of melanocytes. Am J Pathol 123: 195-203
11 Gupta PK, Shurbaji MS, Mintor LJ, Ermatinger SV, Myers J, Quinn TC (1988) Cytopathologic detection of chlamydia trachomatis in vaginopancervical (Fast) smears. Diagn Cytopathol 4: 224-229 12 Hsu SM, Raine L (1984) The use of avidin-biotin-peroxidase complex (ABC) in diagnostic and research pathology. In: De Lellis RA (Ed.) Advances in Immunohistochemistry, pp. 31-42. Year Book Medical Publishers, Chicago 13 Keshgegian AA, Kline TS (1984) Immunoperoxidase demonstration of prostatic acid phosphatase in aspiration biopsy cytology (ABC). Am J Clin Pathol 82: 586-589 14 Kline TS, Lundy J, Lozowski M (1989) Monoclonal antibody B 72.3 - an adjunct for evaluation of suspicious aspiration biopsy cytology from the beast. Cancer 63: 2253-2256 15 Kobayashi TK, Mizuhara S, Arai M, Sawaragi I (1985) Application of immunoperoxidase staining in the cytodiagnosis of herpes simplex keratitis. Diagn Cytopathol 1: 317-321 16 Koelma lA, Nap M., van Steenis GJ, Fleuren GJ (1988) Tumor markers for ovarian cancer: a comparative immunohistochemical and immunocytochemical study of two commercial monoclonal antibodies (OV 632 and OC 125). Am J Clin Pathol
90:391-396
17 Koss LG (1979) Diagnostic Cytology and Its Histopathologic Basis. JB Lippincott CO, Philadelphia. 18 Kurtin PJ, Pinkus GS (1985) Leukocyte common antigen - a diagnostic discriminant between hematopoietic and non-hematopoietic neoplasms in paraffin sections using monoclonal antibodies: Correlation with immunologic studies and ultrastructural localization. Hum Pathol16: 353-365 19 Li CY, Lazcano-Villareal 0, Pierre RV, Yam LT (1988) Immunocytochemical identification of cells in serous effusions. Am J Clin Pathol 88, 696-706 20 Makin CA, Bobrow LG, Bodmer WF (1984) Monoclonal antibody to cytokeratin for use in routine histopathology. J Clin Pathol 37: 975-983 21 Mason M, Bedrossian CWM, Fahey CA (1987) Value of immunocytochemistry in the study of malignant effusions. Diagn Cytopathol 3: 215-221 22 Mukai K, Rosai J (1980) Applications of immunoperoxidase techniques in surgical pathology. In: Fenoglio CM, Wolff M (Eds.) Progress in Surgical Pathology, Vol 1: 115-149. Mason Publishing USA Inc., New York 23 Nadji M (1980) The potential value of immunoperoxidase techniques in diagnostic cytology. Acta Cyrol 24: 442-447 24 Ordonez NG (1989) The immunohistochemical diagnosis of mesothelioma. Differentiation of mesothelioma and lung cancer. Am J Surg Patholl3: 276-291 25 Osamura RY (1989) Applications of immunocytochemistry to diagnostic cytopathology. Diagn Cytopathol 5: 55-63 26 Rajasehar A, Kapila K, Verma K (1989) Value of demonstration of cytokeratin and leukocyte common antigen in poorly differentiated malignant tumors in fine needle aspirates. Acta Cytol 33: 385-389 27 Ramaekers F, Haag D, Jap P, Vooijs PG (1984) Immunochemical demonstration of keratin and vimentin in cytologic aspirates. Acta Cytol 28: 385-392 28 Spagnolo DV, Michie SA, Crabtree GS, Warnke RA, Rou RV (1985) Monoclonal antikeratin (AEI) reactivity in routinely processed tissue from 166 human neoplasms. Am J Clin Pathol 84: 697-704 29 Travis WD, Wold LE (1987) Immunoperoxidase staining of fine needle aspiration specimens previously stained by the Papanicolaou technique. Acta Cytol 31: 517-520 30 Walts AE, Said JW, Shintaku IP (1988) Cytodiagnosis of malignant melanoma-immunoperoxidase staining with HMB-45 antibody as an aid to diagnosis. Am J Clin Pathol 90: 77-80
Immunocytochemistry with Routine Cells . 665 31 Walts AE, Said jW, Shintaku IP, Lloyd RV (1985) Chromogranin as a marker of neuroendocrine cells in cytologic material- an immunocytochemical study. Am J Clin Pathol84: 273-277
32 Warnke RA, Gatter KC, Falini B, Hildreth P, Woolston RE, Pulford K, Cordell JL, Cohen B, De Wolf-Peeters C, Mason DY (1983) Diagnosis of human lymphoma with monoclonal antileukocyte antibodies. N Engl J Med 309: 1275-1281
Received August 2, 1989 . Accepted in revised form January 26, 1990
Key words: Keratin proteins - Leukocyte common antigen - Streptavidin - Immunoperoxidase Jonathan Weintraub, M.D., Centre de cytologie et de depistage du cancer, 51 bd de la Cluse, CH-1205 Geneve, Switzerland
The Application of Immunocytochemical Techniques to Routinely-Fixed and Stained Cytologic Specimens An Aid in the Differential Diagnosis of Undifferentiated Malignant Neoplasms J. Weintraub, M. Redard, D. Wenger and P. Vassilakos Center for Cytology and Cancer Screening and Division of Gynecologic and Obstetric Pathology, Department of Pathology, University of Geneva, Switzerland
SUMMARY
Routinely-fixed and Papanicolaou stained smears with the cytologic diagnosis of undifferentiated malignant neoplasm that had been prepared with cells obtained by fine needle aspiration biopsy (n = 7), pulmonary lavage (n = 5), or thoracentesis (n = 3) from 15 unselected patients were stained by an immunocytochemical technique to evaluate the presence of keratin proteins and the leukocyte common antigen (LCA). Commercially available, well-characterized monoclonal antibodies with specificities for keratin proteins and the leukocyte common antigen, and a streptavidin-biotin-horseradish peroxidase labelling method were used. Evaluation of the stained smears revealed the presence ofone of the two antigens in material obtained from each patient, thus indicating the probable cell-lineage ofthe neoplastic cells. The specificity ofthe monoclonal antibody reagents used was further evaluated in routinely-fixed and stained cytologic material from 24 histologically confirmed carcinomas and 12 lymphomas. In conclusion, immunocytochemical techniques may be successfully applied to routinely processed archival cytologic smears to determine the antigenic profile of morphologically undifferentiated cells and therefore aid in the differential diagnosis of undifferentiated malignant neoplasms.
Introduction Immunocytochemical methods using monoclonal antibodies to identify cell-lineage specific antigens have been increasingly applied in routine histopathology as an aid in the differential diagnosis of undifferentiated malignant neoplasms 3,8,22,32. Among the most commonly employed antibodies are those with specificities for intermediate filament proteins present in the cytoplasm of epithelial and mesenchymal cells 20,28 and for the leukocyte common antigen (LeA), a protein present on the surface of hematopoietic cells 18 ,32. The early identification of the cell-lineage of morphologically undifferentiated malignant cells can be an important element in establishing the definitive diagnosis, in providing prognostic information, 0344-0338/90/0186-0658$3.5010
and in contributing to the choice of appropriate therapy7. Immunocytochemical techniques have been widely applied to cytopathology and numerous studies have shown their utility as a diagnostic aid 1,2,5, 19,23,25,27. These studies have generally been performed using fixed and paraffin embedded cells ("cell blocks") or with cytologic smears prepared using special fixation protocols and preparation procedures. Since there is not always sufficient cytological material to prepare cell blocks, nor to anticipate which samples will present difficult diagnostic problems, some authors have investigated the use of routinelyfixed (ethanol 95%) and stained (Papanicolaou) cytologic smears for immunocytochemistry 1,4,25,29. Several studies have shown that monoclonal or polyclonal antibodies may © 1990 by Gustav Fischer Verlag, Sruttgarr
Immunocytochemistry with Routine Cells . 659
be used to detect specific antigens which remain stable after routine processing. These include intermediate filaments 1, 5, 6, 21, epithelial cell and microbial proteins4, 11, 15,21,29, peptide hormones 9, 23, 29,31, leukocyte antigens and immunoglobulins5,23,29, oncofetal proteins I4 ,21,23,29,30, and prostatic tissue antigens 13 ,23. However, only a limited number of reports, based on small numbers of samples, have evaluated the application of antigen detection techniques to the differential diagnosis of morphologically undifferentiated lesions using routinely fixed and stained materia\2 6 • The present investigation was designed to address 2 related problems, one technical and the other diagnostic. Firstly, we have evaluated the reliability of using routinelyfixed and stained cytologic smears to perform immunocytochemical studies using a limited number of well characterized monoclonal antibodies with specificities for keratin proteins and LCA respectively. Secondly, we have applied this technique to determine the presence of cell-lineage specific antigens in a series of 15 morphologically undifferentiated malignant tumors of unknown origin obtained by cytologic sampling methods. The presence of multiple antigens was evaluated simultaneouslyon the same slide.
Material and Methods
Patient Samples Routinely-fixed (ethanol 95% - ether, 1: 1 v/v) and stained (method of Papanicolaou) smears collected during the period 1986-1988 were retrieved from the archives of the Cytology Center and from the Laboratory Clinic, Geneva, Switzerland. The samples had been obtained by fine needle aspiration biopsy (n = 7), pulmonary or peritoneal lavage (n = 5), or thoracentesis (n = 3). Smears were collected from 15 cases with the cytologic diagnosis of undifferentiated malignant neoplasm (Table 1). In all cases a subsequent histopathological examination was performed. Controls for monoclonal antibody specificity included routinely-fixed and stained smears from 24 patients with the cytologic diagnosis of carcinoma (Table 2) and from 12 with the cytological diagnosis of malignant lymphoma (Table 3). All diagnoses were histologically confirmed.
Antibody Reagents and Immunocytochemical Methods The antibodies used in this study, the appropriate dilutions and the respective source of supply are listed in Table 4. For immunocytochemical staining a peroxidase conjugated streptavidin labelling procedure was used12, 19. The details of the staining protocol are shown in Table 5. No specific destaining step was employed to remove the Papanicolaou stain. The cytologic material was subdivided into 3 regions in order to stain simultaneously for several antigens on the same slide. Following removal of the coverslip, a band-like zone, 0.3 cm in width, situated between the different regions, was scraped clean of cytologic material. In this manner, and using a careful technique, different primary antibodies placed on different areas of the slide could be kept from mixing. No additional products, such as mounting medium, were necessary to separate the different regions.
Controls and the Evaluation of Immunostaining A negative control was included with each patient sample studied by omitting the primary antibody (and replacing it with phosphate buffered saline, pH 7.2, containing 5% bovine serum albumin - PBS-5 01o BSA). Readily identifiable benign epithelial and lymphoid elements served as internal positive and negative controls when present in the cytologic preparations. Positive cytoplasmic staining was interpreted as demonstrating the presence of keratin. In contrast, staining of cells for LCA was considered positive when the distribution of labelling was primarily membranous. An estimation of the distribution (proportion of stained neoplastic cells) and intensity of immunostaining (weak, moderate, strong) was performed by classifying at least 200 consecutive neoplastic cells in contiguous fields (when sufficient cytologic material was available). The evaluation of the results of the immunostaining of cases of undifferentiated malignant neoplasm was performed without prior knowledge of the histologic diagnosis. The evaluation of the control series of carcinomas and lymphomas was done by an observer OW) with no knowledge of the cytologic or histopathologic diagnosis. The diagnosis in all cases was histologically confirmed. Cytologic diagnoses were based on standard criteria 17.
Results A total of 15 specimens with the cytologic diagnosis of undifferentiated malignant neoplasm were examined by immunocytochemistry for the presence of keratin proteins and the leukocyte common antigen. In all cases the identification of the product of the staining reaction was unambiguous and in the appropriate cellular localization, i.e. the distribution of staining for keratin was cytoplasmic and for LCA was membranous and cytoplasmic (Fig. 1). Background staining, as a result of non-specific binding was minimal. The results of the immunostaining are presented in Table 1. The antigenic profile of the atypical cells was compatible with an epithelial cell-lineage and the diagnosis of carcinoma in 12 cases and with a leukocyte cell-lineage and the diagnosis of lymphoma in 3 cases. At least 25% of the neoplastic cells were stained in all cases. The staining was moderate to strong. One case (pt 1) showed a punctate pattern of intracytoplasmic staining. In all cases the subsequent histologic diagnosis confirmed the interpretation of the immunocytochemical staining pattern. In this series, the most frequent diagnostic dilemma concerned the distinction between lymphoma and small cell anaplastic carcinoma and, secondly, between large cell carcinoma and immunoblastic lymphoma. In an effort to control the specificity and reliability of the antibody staining reagents, the antigenic profile of neoplastic cells of known origin was determined in a selected series of cases in which the cytologic and histologic diagnoses were available. These included 24 cases of carcinoma and 12 cases of lymphoma. There were 8 small cell carcinomas of the lung, 12 adenocarcinomas from prostate, breast, ovary, stomach and pancreas, 1 hepatocellular carcinoma, 2 squamous cell carcinomas, and 1 transitional cell carcinoma of the bladder (Table 2). The 12 lymphomas included 6 centrocyticlcentroblastic, or well differentiated lymphocytic types, 4 immunoblastic
660 . Weintraub,
J. et a!.
Table 1. Clinico-pathologic features and immunostaining characteristics for 15 cases with the initial cytologic diagnosis of undifferentiated malignant neoplasm Sex/age
Source of cytologic sample
Results of immunostaining Percent positive Definitive histologic diagnosis keratin cells/staining intensity AE l/AE 3 CAM 5.2 LCA
1 2
F/80 Ml73
pulmonary lavage FNABal-iliac fossa
+
+
3 4 5 6
F/65 F/55 F/34 F/64
pulmonary lavage thoracentesis pulmonary lavage pulmonary lavage
+ + + +
+ + + +
50%/++ 100%/+++ 100%/+++ rare cells/+ + +
7
Ml80
FNAB-Iymph node
+
+
100%/++ - +++
8 9
Ml79 Ml61
FNAB-Iung thoracentesis
+ +
+ +
75%/++ 100%/++
10 11
F/64 F/77
peritoneal lavage FNAB-lymph node
+
+
12
Ml74
thoracentesis
13
F/85
+
75%/++
14
Ml74
FNAB-subcutaneous + nodule FNAB-salivary gland +
+
100%/+++
15
Ml69
FNAB-Iymph node
+
100%/+++
Patient number
+
+
75%1++ 25%/+
+
50% 75%/++
+
100%/++
small cell carcinoma of the lung malignant lymphoma-centrocytic type small cell carcinoma of the lung undifferentiated carcinoma pneumoblastoma large cell undifferentiated carcinoma of the lung small cell carcinoma of the bladder small cell carcinoma of the lung poorly differentiated squamous cell carcinoma of the lung endometrial adenocarcinoma malignant lymphoma-B-immunoblastic malignant lymphoma-centroblastic metastatic ductal carcinoma of the breast large cell undifferentiated carcinoma of the lung small cell carcinoma of the lung
a) FNAB: fine needle aspiration biopsy.
Table 2. Clinico-pathologic features and immunostaining characteristics for "known" cases of carcinoma Sex/age
Source of cytologic sample
Keratin % positive cells/staining intensity
Definitive histologic diagnosis
1 2
Ml76 Ml53
pulmonary lavage pulmonary lavage
100%/++ rare neop!. cells/+++
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
F/60 Ml70
pulmonary lavage thoracentesis thoracentesis FNAB-prostate FNAB-prostate FNAB-lung paracentesis FNAB-liver FNAB-Iymph node paracentesis pulmonary lavage pulmonary lavage pulmonary lavage pulmonary lavage thoracentesis thoracentesis peritoneal lavage thoracentesis thoracentesis paracentesis paracentesis thoracentesis
rare neop!. cellsl+ + 50%/++ 50%1++ 25%1+ 25%/+ 50%/++ 100%/++ 75%1++ 50%/++ 90%1++ 75%/+ 50%/+ 50%/+ 50%1+ 100%/+++ 50%1++ 100%/++ - +++ 50%/++ 100%1+++ 100%/++ 100%/++ 100%/+++
small cell carcinoma of the lung small cell and poorly differentiated squamous cell carcinoma of the lung poorly differentiated squamous cell carcinoma of the lung small cell carcinoma of the lung small cell carcinoma of the lung adenocarcinoma of the prostate adenocarcinoma of the prostate small cell carcinoma of the lung papillary transitional carcinoma of the bladder hepatocellular carcinoma infiltrating ductal carcinoma of the breast adenocarcinoma small cell carcinoma of the lung small cell carcinoma of the lung small cell carcinoma of the lung poorly differentiated squamous cell carcinoma infiltrating ductal carcinoma of the breast papillary serous carcinoma of the ovary poorly differentiated ovarian carcinoma infiltrating ductal carcinoma of the breast infiltrating ductal carcinoma of the breast papillary serous cystadenocarcinoma of the ovary moderately differentiated gastric adenocarcinoma infiltrating ductal carcinoma of the breast
Patient number
22
23 24
Min
Ml81 Ml86 Ml79 F/74 Ml76 F/42 F/43 Ml89 Ml75 Ml69 Ml77 F/58 F/64 F/64 F/68 F/73 F/75 Ml60 F/57
Immunocytochemistry with Routine Cells . 661
A
c Fig. 1. Cell smears from 2 lesions with the cytologic diagnosis of undifferentiated malignant neoplasm stained by immunocytochemistry (original magnification x 1070) (Table 1). - A: Patient number 7: neoplastic cells stained for keratin proteins (definitive histologic diagnosis - carcinoma). - B: Patient number 7: neoplastic cells stained for LCA - note positively stained macrophage. - C: Patient number 12: cells stained for keratin; positive cytoplasmic staining is present in 2 mesothelial cells (definitive histologic diagnosis lymphoma). - 0: Patient number 12: Cells stained for LCA; all cells show membrane and some cytoplasmic staining with the exception of 2 mesothelial cells.
662 . Weintraub, J. et al.
Fig. 2. Cell smears from "known" cases of carcinoma and lymphoma stained by immunocytochemistry (original magnification x 1000) (Table 2 and 3). - A: Patient number 8 (Table 2): neoplastic cell stained for keratin proteins - mature lymphocytes are unstained (definitive histologic diagnosis - carcinoma). - B: Patient number 8 (Table 2): neoplastic cells stained for LCA-positive staining is limited to lymphocytes. - C: Patient number 2 (Table 3): neoplastic cells stained for keratin - one mesothelial cell shows cytoplasmic staining (definitive histologic diagnosis -lymphoma). D: Patient number 2 (Table 3): neoplastic cells are uniformly stained for LCA-note absence of staining in one mesothelial cell.
Immunocytochemistry with Routine Cells . 663
types, and 1 case each of multiple myeloma and mixedcellularity Hodgkin's disease (Table 3). The quality of the staining reactions was identical to that observed in the series of morphologically undifferentiated malignant neoplasms. No cases showed simultaneous staining for lymphoid and epithelial antigens and there was no background or nonspecific staining to complicate the evaluation of the immunostaining. All carcinomas stained positively for keratin; there was no staining with LCA. The distribution and intensity of staining varied (Table 1). The lymphomas were uniformly positive for LCA with between 90%-100% of the neoplastic cells stained. The intensity varied from moderate to strong (data not shown). As previously noted, staining for LCA was pronounced along the cell surface, while staining for keratin was cytoplasmic (Fig. 2).
The results obtained in this study confirm and extend previous reports in which the presence of specific antigens in cells present on routinely-fixed and stained smears was evaluated 1,21,29. While the immunostaining protocol used in this investigation was based on a traditional peroxidase labelling procedure, several important technical differences exist with previously reported protocols 29 . These include: 1) the elimination of a specific destaining step which was found to be unnecessary; 2) the use of a streptavidin-biotin-horseradish peroxidase-diamino benzidine labelling system, which virtually eliminated nonspecific background staining; 3) the use of well-characterized and commercially available monoclonal antibody diagnostic reagents to improve specificity of staining; and 4) the ability to stain for several antigens simultaneously
Table 3. Clinico-pathologic features and immunostaining characteristics for "known" cases of malignant lymphoma
1
2 3 4 5
6 7 8 9 10 11
12
1
Sexlage
Source of cytologic Definitive histologic sample diagnosis with subclassification
Ml85 Ml82 Ml63 Ml85 Ml33 F/85 F/83 FI50
paracentesis thoracentesis paracentesis FNABLJymph node FNAB-lymph node paracentesis thoracentesis thoracentesis thoracentesis thoracentesis thoracentesis thoracentesis
Ml49 F/80 F/71
Ml70
Antibody (antigen)
Cellular reactivity
AE lIAE 3 (keratin) CAM 5.2 (keratin)
epithelial tumors epithelial tumors
Dilution sourceI
1: 50 Hybritech 1: 50 Amersham anti-LCA (leukocyte neoplastic leukocytes 1: 20 Dako common antigen, CD 45) 1 : 100 Amerbiotinylated rabbit anti-mouse Ig sham I Hybritech, San Diego CA; Amersham, U.K.; Dako, Glostrup, Denmark.
Table 5. Immunostaining protocol
1. remove coverslip with xylol
Discussion
Patient number
Table 4. Antibodies used in the analysis of cytologic smears
FNAB-fine needle aspiration biopsy.
multiple myeloma well differentiated lymphocytic unclassified centrocytic-centroblastic Hodgkin's disease mixed cellularity centrocytic T-lymphocytic T-Iymphocytic centroblastic centrocytic-centroblastic T-Iymphocytic centrocytic
2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15.
place in 95% ethanol, 2 x 1 minI incubate in methanol- 3% H 20 2, 10 min rehydrate cells in a graded series of ethanols place in PBS2, 5 min incubate in PBS - 5% BSA2, 10 min incubate with specific primary monoclonal antibody, overnight, 4°C place in PBS,S min incubate with biotinylated rabbit anti-mouse Ig2, diluted 1 : 100,30 min place in PBS,S min incubate with streptavidin-biotin-horseradish peroxidase complex 3 diluted 1 : 200, 15 min place in PBS,S min incubate with diaminobenzidine solution4, 5 min rinse with distilled water counterstain and coverslip
I All steps were performed at 20 °c unless otherwise specified. 2 Abbreviations used: PBS, phosphate buffered saline pH 7.2; BSA, bovine serum albumin; Ig, immunoglobulin. - 3 The streptavidin-biotin-horseradish peroxidase complex was obtained from Amersham, U.K. - 4 50 mg diaminobenzidinellOO ml distilled water containing 3% H 20 2.
on the same smear in order to conserve material and to diminish problems resulting from the uneven distribution of neoplastic cells from smear to smear. In addition, little cell loss by detachment from slides was noted during the immunocytochemical staining procedure. Antigenicity, in the case of the antigens evaluated, was maintained for months and, in some cases, years. As a result of these factors, the immunocytochemical signal was clearly evident and easily evaluable; it was uncomplicated by non-specific background staining. This investigation focused on the application of immunocytochemical methods to the differential diagnosis of morphologically undifferentiated malignant neoplasms. Fortuitously, the series of 15 unselected patients studied did not include any in whom the diagnosis of sarcoma or melanoma was subsequently confirmed histopathologically. However, it is clear that these diagnoses and others
664 . Weintraub, J. et al.
must be included in the differential diagnosis of morphologically undifferentiated malignant neoplasms. Further, antibody reagents should be employed which permit the identification of the corresponding cell-lineage. The results of preliminary studies in our laboratory have confirmed the utility of broadening the antibody panel by using reagents specific for the intermediate filament protein, vimentin, and the melanoma associated antigen, HMB45 10,3°. These well-characterized and commercially available monoclonal antibody reagents give a similar quality of labelling as described in this study for antikeratin and anti-LeA antibodies. Once an epithelial cell lineage has been determined, the antibody panel may be broadened still further to determine the organ of origin of metastatic carcinomatous cells. Preliminary investigations have confirmed the utility, in specific clinical situations, of incorporating monoclonal antibody reagents with specificities for prostatic and ovarian antigens 13 ,16. However, our experience with epithelial cell associated antigens has been mixed and the specificity, using routinely fixed and stained material, of some of the antibodies remains to be established 24 • In conclusion, immunocytochemical staining methods may be applied to archival routinely-fixed and stained cell smears in an effort to resolve diagnostic problems.
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17 Koss LG (1979) Diagnostic Cytology and Its Histopathologic Basis. JB Lippincott CO, Philadelphia. 18 Kurtin PJ, Pinkus GS (1985) Leukocyte common antigen - a diagnostic discriminant between hematopoietic and non-hematopoietic neoplasms in paraffin sections using monoclonal antibodies: Correlation with immunologic studies and ultrastructural localization. Hum Pathol16: 353-365 19 Li CY, Lazcano-Villareal 0, Pierre RV, Yam LT (1988) Immunocytochemical identification of cells in serous effusions. Am J Clin Pathol 88, 696-706 20 Makin CA, Bobrow LG, Bodmer WF (1984) Monoclonal antibody to cytokeratin for use in routine histopathology. J Clin Pathol 37: 975-983 21 Mason M, Bedrossian CWM, Fahey CA (1987) Value of immunocytochemistry in the study of malignant effusions. Diagn Cytopathol 3: 215-221 22 Mukai K, Rosai J (1980) Applications of immunoperoxidase techniques in surgical pathology. In: Fenoglio CM, Wolff M (Eds.) Progress in Surgical Pathology, Vol 1: 115-149. Mason Publishing USA Inc., New York 23 Nadji M (1980) The potential value of immunoperoxidase techniques in diagnostic cytology. Acta Cyrol 24: 442-447 24 Ordonez NG (1989) The immunohistochemical diagnosis of mesothelioma. Differentiation of mesothelioma and lung cancer. Am J Surg Patholl3: 276-291 25 Osamura RY (1989) Applications of immunocytochemistry to diagnostic cytopathology. Diagn Cytopathol 5: 55-63 26 Rajasehar A, Kapila K, Verma K (1989) Value of demonstration of cytokeratin and leukocyte common antigen in poorly differentiated malignant tumors in fine needle aspirates. Acta Cytol 33: 385-389 27 Ramaekers F, Haag D, Jap P, Vooijs PG (1984) Immunochemical demonstration of keratin and vimentin in cytologic aspirates. Acta Cytol 28: 385-392 28 Spagnolo DV, Michie SA, Crabtree GS, Warnke RA, Rou RV (1985) Monoclonal antikeratin (AEI) reactivity in routinely processed tissue from 166 human neoplasms. Am J Clin Pathol 84: 697-704 29 Travis WD, Wold LE (1987) Immunoperoxidase staining of fine needle aspiration specimens previously stained by the Papanicolaou technique. Acta Cytol 31: 517-520 30 Walts AE, Said JW, Shintaku IP (1988) Cytodiagnosis of malignant melanoma-immunoperoxidase staining with HMB-45 antibody as an aid to diagnosis. Am J Clin Pathol 90: 77-80
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32 Warnke RA, Gatter KC, Falini B, Hildreth P, Woolston RE, Pulford K, Cordell JL, Cohen B, De Wolf-Peeters C, Mason DY (1983) Diagnosis of human lymphoma with monoclonal antileukocyte antibodies. N Engl J Med 309: 1275-1281
Received August 2, 1989 . Accepted in revised form January 26, 1990
Key words: Keratin proteins - Leukocyte common antigen - Streptavidin - Immunoperoxidase Jonathan Weintraub, M.D., Centre de cytologie et de depistage du cancer, 51 bd de la Cluse, CH-1205 Geneve, Switzerland
