Thorax: first published as 10.1136/thx.40.2.91 on 1 February 1985. Downloaded from Thorax 1985;40:91-95

Comparison of tumour markers in malignant and pulmonary adenocarcinoma

AR GIBBS, R HARACH, JC WAGNER, B JASANI From the Pathology Department, Welsh National School, Cardiff, and the Medical Research Council Pneumoconiosis Unit, Llandough Hospital, Penarth, Glamorgan

ABSTRACr Immunohistological methods were used to investigate the presence of carcinoem- bryonic antigen, ,31 pregnancy specific glycoprotein, /8 subunit of human chorionic gonado- trophin, human placental lactogen, calcitonin, and keratin in formalin fixed tissue from 29 malig- nant and 27 pulmonary adenocarcinomas. Malignant mesotheliomas were nega- tive for tumour markers except for the ,8 subunit of human chorionic gonadotrophin and keratin, one and 13 cases respectively being positive for these. Pulmonary adenocarcinomas, however, were frequently positive for tumour markers-namely, carcinoembryonic antigen (24), ,3B preg- nancy specific glycoprotein (23), ,8 subunit of human chorionic gonadotrophin (8), human placen- tal lactogen (2), calcitonin (3), and keratin (12). The presence of carcinoembryonic antigen and ,/3 pregnancy specific glycoprotein within an intrathoracic tumour is strong evidence against its being of mesothelial origin. copyright. The accurate diagnosis of malignant tumours is an needs to be recognised well in advance, so that the essential part of the management of patients, but for appropriate fixation and embedding techniques can some tumours it can still pose many problems. One be used. particularly difficult and important problem is the The introduction of immunocytochemical techni- separation of malignant mesothelioma from pulmo- ques into routine pathological diagnosis has led to http://thorax.bmj.com/ nary adenocarcinoma-difficult because these improvement in tumour diagnosis generally; in the tumours may closely resemble each other in their differential diagnosis of malignant mesothelioma pattern of spread and their microscopic appear- and adenocarcinoma reports have been conflicting. ances,' 2 and important not only for clinical man- Antibodies to carcinoembryonic antigen and keratin agement but also for reasons of industrial compensa- have been used, and it has been claimed that car- tion. cinoembryonic antigen is frequently present in pul- Many attempts have been made to provide an eas- monary adenocarcinoma but almost invariably ily used technique which gives a clear separation absent in malignant mesothelioma.6-9 Keratin pro- on September 30, 2021 by guest. Protected between the two tumours. Mucin histochemistry has teins are found by some workers to be frequently been used, positive staining of tumour cells for present in malignant mesothelioma and absent in hyaluronic acid favours a diagnosis of malignant pulmonary adenocarcinoma,9 10 while others have mesothelioma, whereas the presence of neutral found positive keratin staining in pulmonary mucin is indicative of adenocarcinoma.34 This tech- adenocarcinoma also." 12 nique is not, however, widely used, partly because In a previous study of tumours using not every adenocarcinoma contains neutral mucin, immunocytochemical techniques we studied various but also because the preservation of intracellular antigens and found that immunoreactive car- hyaluronic acid requires special fixative methods. cinoembryonic antigen was present in 71%, /,3 Ultrastructural features can also be used to distin- pregnancy specific glycoprotein (SP,) in 68%, ,3 guish these two tumours.5 Here too the problem subunit of human chorionic gonadotrophin (,3HCG) in 36%, human placental lactogen in 20%, and cal- Address for reprint requests: Dr AR Gibbs, Pathology Depart- citonin in 14%.'3 Because of the conflicting results ment, Welsh National School of Medicine, Cardiff CF4 4XN. of other studies in the separation of pulmonary adenocarcinoma and mesothelioma, we have Accepted 3 September 1984 applied these five antibodies together with a keratin 91 Thorax: first published as 10.1136/thx.40.2.91 on 1 February 1985. Downloaded from

92 Gibbs, Harach, Wagner, Jasani antibody to a new series of 56 pulmonary tumours to gonadotrophin, and human placental lactogen. define which investigations are likely to be of value Trypsinisation was not performed for carcino- in this important differential diagnosis. embryonic antigen or keratin. Appropriate positive and negative controls were included with each batch Methods of staining. The dilutions and incubation times were selected as those which gave consistently good stain- Sections (5 ,um thick) were taken from formalin ing with a clear background in an appropriate positive fixed, conventionally processed paraffin embedded control tissue. These were slightly modified from material. Twenty nine malignant mesotheliomas and those used in our previous study for consistency and 27 pulmonary adenocarcinomas were studied. The greater reliability.'3 malignant mesotheliomas were all from necropsy The degree of staining for the appropriate antigen cases with typical macroscopic and microscopic was graded for each tumour as follows: 0-negative; appearances and without any other probable prim- + (weak)-less than 5% of the tumour cells ary source; they comprised 12 purely epithelial intensely or moderately stained; + + (moderate)- tumours, 14 mixed epithelial and sarcomatous 5-33% of the tumour cells intensely or moderately tumours, and three purely sarcomatous tumours. stained; + + + (strong)-more than 33% of the Histochemical staining for neutral mucin (periodic- tumour cells intensely or moderately stained. acid-Schiff diastase technique'4) was negative for each malignant mesothelioma. The pulmonary Results adenocarcinomas were from both surgical (13) and necropsy (14) cases: six were well differentiated, The results of staining for each tumour marker are three moderately differentiated, and 11 poorly dif- shown in the table. Carcinoembryonic antigen was ferentiated; three were solid tumours with mucin present in 24 out of 27 adenocarcinomas tested but content, one was a bronchioloalveolar , absent in all the malignant mesotheliomas. The and three were adenosquamous . tumour product of diaminobenzidine was located copyright. The six immunohistological markers were local- predominantly along the luminal surfaces of the ised by a modification of the DPN-hapten staining tumour cells but it was also seen to a lesser extent in techniques,'5 16 the following dilutions and incuba- both diffuse and granular patterns within the cyto- tion times being used: (1) carcinoembryonic antigen plasm of the tumour cells, particularly in the less (Dakopatts), 1:2000 for 15 hours; (2) ,f3 pregnancy well differentiated adenocarcinomas, but this was specific glycoprotein (Dakopatts), 1:1600 for 15 not statistically significant. Two of the three adeno- http://thorax.bmj.com/ hours; (3) keratin (Miles-Yeda), 1:200 for 15 hours; squamous carcinomas showed moderate staining (4) 18 subunit of human chorionic gonadotrophin for carcinoembryonic antigen; there was staining of (Dakopatts), 1:400 for 15 hours; (5) human placen- both the glandular and the squamous elements, the tal lactogen (Dakopatts), 1:800 for 15 hours; (6) latter being mainly located along cell membranes calcitonin 1:200 for 15 hours. and within epithelial pearls. Prior trypsinisation'7 was carried out for one hour Staining for 13, pregnancy specific glycoprotein for calcitonin and four hours for 1, pregnancy was positive in 23 of 27 adenocarcinomas but nega- the subunit of human chorionic specific glycoprotein, ,8 tive in all malignant mesotheliomas tested. There on September 30, 2021 by guest. Protected

Results ofimmunostaining for the six tumour markers Tumour Histological Degree ofpositvity* Total marker type of tumour 0 + ++ +++ Carcinoembryonic antigen Adenocarcinoma 3 8 5 11 27 Mesothelioma 29 0 0 0 29 ,(, pregnancy specific glycoprotein Adenocarcinoma 4 10 7 6 27 Mesothelioma 29 0 0 0 29 The beta subunit of human chorionic Adenocarcinoma 17 8 0 0 25 gonadotrophin Mesothelioma 26 1 0 0 27 Human placental lactogen Adenocarcinoma 23 2 0 0 25 Mesothelioma 27 0 0 0 27 Calcitonin Adenocarcinoma 22 3 0 0 25 Mesothelioma 27 0 0 0 27 Keratin Adenocarcinoma 15 7 3 2 27 Mesothelioma 16 8 2 3 29 *(}negative; +-<5% of tumour cells intensely or moderately stained; ++-5-33% of cells intensely or moderately stained; + + +->33% of cells intensely or moderately stained. Thorax: first published as 10.1136/thx.40.2.91 on 1 February 1985. Downloaded from Comparison of tumour markers in malignant mesothelioma and pulmonary adenocarcinoma 93 was no correlation between the histological differen- gators. Wang et a16 found carcinoembryonic antigen tiation of the adenocarcinomas and the degree of positivity in all 12 pulmonary adenocarcinomas positivity. The pattern of staining was similar to that tested but in none of nine malignant mesotheliomas. of carcinoembryonic antigen, being strongest along Whitaker et al' found positivity in 22 of 26 pul- the luminal border of the cells but also present monary adenocarcinomas and in none of 43 within the cytoplasm in diffuse and granular pat- malignant mesotheliomas. Corson and Pinkus,8 terns. Two of the three adenosquamous carcinomas however, found weak staining in eight and moderate showed weak staining in both the glandular and the staining in one of 20 malignant mesotheliomas, squamous components. The latter showed staining whereas they found that all adenocarcinomas ex- of the cell borders and epithelial pearls. hibited strong or moderate staining. Pascal et al'8 It is noteworthy that staining for either car- found positivity in 16 of 22 adenocarcinomas. We cinoembryonic antigen or fl, pregnancy specific conclude that a positive immunoreaction for car- glycoprotein or both was positive in all adeno- cinoembryonic antigen is strong evidence against a carcinomas but was negative for both in all malig- mesothelial origin for a primary intrathoracic nant mesotheliomas. Weak positive staining for the tumour. f3 subunit of human chorionic gonadotrophin was The so called trophoblast specific pregnancy observed in eight of 25 adenocarcinomas and one of proteins-/3, pregnancy specific glycoprotein, the 27 malignant mesotheliomas. The staining was pre- beta subunit of human chorionic gonadotrophin and dominantly intracytoplasmic in diffuse or granular human placental lactogen, normally synthesised by patterns. the human placenta-have now been demonstrated Weak positive diffuse intracytoplasmic staining by immunolocalisation procedures in tissue sections for human placental lactogen was seen in two of 25 of certain non-trophoblastic as well as trophoblastic adenocarcinomas, while it was negative in all 27 tumours.'3 1920 A few studies have reported raised malignant mesothelioma. serum concentrations of various placental proteins Staining for calcitonin was weakly positive in in a small proportion of patients with lung car- three of 25 adenocarcinomas; the three positives cinomas.21 23 Wilson et a124 investigated the beta copyright. comprised a solid carcinoma with mucin content, a subunit of human chorionic gonadotrophin in tissue poorly differentiated acinar adenocarcinoma, and a sections from 61 lung tumours and found positive moderately differentiated papillary adenocar- staining in 84%. In a previous study using immuno- cinoma. It was negative in all 27 malignant localisation procedures we showed /,3 pregnancy mesotheliomas. specific glycoprotein in 68%, the beta subunit of Positive staining for keratin was observed in 12 of human chorionic gonadotrophin in 36%, and http://thorax.bmj.com/ 27 adenocarcinomas and in 13 of 29 malignant placental lactogen in 20%.'3 The present study of a mesotheliomas, but there was no direct correlation separate group of tumours shows that pulmonary with differentiation. In the adenocarcinomas stain- adenocarcinomas are frequently positive for f,3 ing was seen predominantly at the periphery of the pregnancy specific glycoprotein (85%), less cells. The malignant mesotheliomas showed mainly frequently for the beta subunit of human chorionic diffuse intracytoplasmic staining, present in both gonadotrophin (32%), and positive for placental epithelial and sarcomatous areas but more prevalent lactogen in only 12%. There was no direct relation- in the former. ship between the presence of one placental protein on September 30, 2021 by guest. Protected There appeared to be no significant differences in and the presence of another. With the exception of a the staining of any one marker between necropsy case of that showed weak staining for the beta sub- and surgical tissues. unit of human chorionic gonadotrophin, malignant mesotheliomas appeared uniformly negative for Discussion each of the placental proteins studied. Immuno- histological investigation of placental proteins in Our study clearly shows that the use of immuno- malignant mesotheliomas has not previously been localisation procedures for certain antigens is a val- reported and our results show that the presence of a uable adjunct in the differential diagnosis of malig- positive immunoreaction of 8, pregnancy specific nant mesothelioma from pulmonary adenocar- glycoprotein is again strong evidence against a cinoma. The most useful in this respect appear to be mesothelial origin for a primary intrathoracic carcinoembryonic antigen and f3, pregnancy specific tumour. glycoprotein. Raised levels of calcitonin have been described in Our findings of carcinoembryonic antigen in 89% various lung tumours25-26 but few immuno- of adenocarcinomas and in none of the malignant histological studies have reported. Calcitonin posi- mesotheliomas accords with those of other investi- tive cells have been described in some bronchial car- Thorax: first published as 10.1136/thx.40.2.91 on 1 February 1985. Downloaded from

94 Gibbs, Harach, Wagner, Jasani cinoids, small cell carcinomas, and adenocarcinomas of malignant mesothelioma if the histological of lung.'3 27-28 The present study showed weak appearances are compatible. Since antibodies to positivity in 12% of pulmonary adenocarcinomas. both carcinoembryonic antigen and t3, pregnancy All malignant mesotheliomas were negative. specific glycoprotein are readily available as stand- Previous results for keratin staining in adenocar- ardised commercial products, the combined cinomas have been conflicting. Corson and Pinkus8 immunolocalisation of carcinoembryonic antigen stated that staining for keratin was weak or negative and the I,3 pregnancy specific glycoprotein should in 18/20 pulmonary adenocarcinomas and positive prove an efficient routine means of differentiating in all 20 malignant mesotheliomas studied. Other lung adenocarcinoma from difficult cases of pleural workers, however, have reported keratin positivity mesothelioma in both necropsy and surgical cases. in appreciable proportion of adenocarcinomas.'22930 Our results show keratin positivity as frequently in We gratefully acknowledge the assistance of Profes- and of Miss J adenocarcinomas (12/27) as in malignant sor ED Williams and Mr ND Thomas, mesotheliomas (13/29). The frequency of keratin Stitfall for typing the manuscript. positivity in our series of malignant mesotheliomas References is less than in most other series.8 This could be accounted for in several ways. It is possible that 1 Harwood TR, Gracey DR, Yakoo H. Pseudo- tumour keratin antigenicity might be lost after death mesotheliomatous carcinoma of the lung. A variant of peripheral lung . Am J Clin Pathol 1976; so that our postmortem series might be expected to 65:159-67. show a lower incidence of positivity than the 2 McCaughey WTE, Oldham PD. Diffuse meso- reported series using surgical pathological speci- theliomas: observer variation in histological diagnosis. mens. We found no difference, however, in the inci- In: Bogovski P, Gilson JC, Timbrell V, Wagner JC, dence of keratin positivity between postmortem and eds. Biological effects ofasbestos. Lyons: International surgical cases of adenocarcinoma. Secondly, some Agency for Research on Cancer, 1973:58-61. workers have reported better results for keratin 3 Wagner JC, Munday DE, Harrington JS. Histochemi-

cal demonstration of hyaluronic acid in pleuralcopyright. staining after fixation in ethanol, methanol, or mesotheliomas. J Pathol Bacteriol 1962;84:73-8. Carnoy's solution.'2 Thirdly, results for keratin 4 Kannerstein M, Churg J, Magner D. Histochemical positivity could vary because cytokeratins are studies in the diagnosis of mesothelioma. In: Bogovski heterogeneous and different antibodies have differ- P, Gilson JC, Timbrell V, Wagner JC, eds. Biological ent against different cytokeratin effects of asbestos. Lyons: International Agency for specificities Research on Cancer, 1973:62-4. polypeptides.3' More work is needed with mono- 5 Wang NS. Electron microscopy in the diagnosis of http://thorax.bmj.com/ clonal antibodies to determine whether some are pleural mesotheliomas. Cancer 1973;31:1046-54. more useful than others in differentiating malignant 6 Wang N, Huang S, Gold P. Absence of carcinoem- mesothelioma from pulmonary adenocarcinoma. bryonic antigen-like material in mesothelioma. An The alcian blue staining technique with and with- immunohistochemical differentiation from other lung . Cancer 1979;44:937-43. out hyaluronidase was not used with these tumours 7 Whitaker D, Sterrett GP, Shilkin KB. Detection of tis- because we, like others,32 have found that after for- sue CEA-like substance as an aid in the differential malin fixation hyaluronic acid can be demonstrated diagnosis of malignant mesothelioma. Pathology in less than half of malignant mesotheliomas. The 1982; 14:255-8. malignant mesotheliomas chosen for this series were 8 Corson JM, Pinkus GA. Mesothelioma: Profile of on September 30, 2021 by guest. Protected An typical in gross and light microscopical appearance keratin proteins and carcinoembryonic antigen. immunoperoxidase study of 20 cases and comparison with little doubt as the the diagnosis. with pulmonary adenocarcinoma. Am J Pathol Our study indicates that of the six tumour markers 1982; 108:80-7. investigated carcinoembryonic antigen and 8, pre- 9 Said JW, Nash G, Tepper G, Banks-Schlegel S. Kera- gnancy specific glycoprotein are the most helpful in tin proteins and carcinoembryonic antigen in lung car- the distinction of malignant mesothelioma from cinoma. Hum Pathol 1983;14:70-6. 10 Schlegel R, Banks-Schlegel S, McLeod JA, Pinkus GS. pulmonary adenocarcinoma. Carcinoembryonic Immunoperoxidase localisation of keratin in human antigen and the f,3 pregnancy specific glycoprotein . Am J Pathol 1980; 101:41-50. were negative in all the malignant mesotheliomas 11 Espinoza CG, Azar HA. Immunohistochemical local- tested whereas one or both were present in all the isation of keratin-type proteins in epithelial neoplasms. adenocarcinomas. Thus a positive result for either Am J Clin Pathol 1982;78:500-7. or pregnancy specific 12 Ramaekers F, Puts J, Maester 0, Kant A, Jap P, carcinoembryonic antigen f,3 Vooijs P. Demonstration of keratin in human adeno- glycoprotein in a tumour is strong evidence against a carcinoma. Am J Pathol 1983;111:213-23. diagnosis of malignant mesothelioma. A negative 13 Harach HR, Skinner M, Gibbs AR. Biological markers result for both carcinoembryonic antigen and f, in human lung carcinoma: an immunopathological pregnancy specific glycoprotein is highly suggestive study of six antigens. Thorax 1983;38:937-41. Thorax: first published as 10.1136/thx.40.2.91 on 1 February 1985. Downloaded from

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1984; 114:9-17. copyright. 23 Grudzinskas JS, Coombes RC, Ratcliffe JG, Gordon 32 Whitaker D, Shilkin KB. Diagnosis of pleural malig- YB, Powles TJ, Munro Neville A, Chard T. Circulating nant mesothelioma in life-a practical approach. J levels of pregnancy specific (3, glycoprotein in patients Pathol 1984; 143:147-75. http://thorax.bmj.com/ on September 30, 2021 by guest. Protected