Académie internationale de Pathologie - Division arabe XX ème congrès novembre 2008 Alger. Immunohistochemistry in malignant mesotheliomas

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1 Académie internationale de Pathologie - Division arabe XX ème congrès novembre 2008 Alger Immunohistochemistry in malignant mesotheliomas Françoise Thivolet-Béjui Groupement Hospitalier Est Lyon-Bron Université Cl Bernard Lyon1

2 WHO 2004: Mesothelial tumours Diffuse malignant mesothelioma MM Epithelioid mesothelioma Sarcomatoid mesothelioma Desmoplastic mesothelioma Biphasic mesothelioma Localized malignant mesothelioma Other tumours of mesothelial origin Well differentiated papillary mesothelioma Adenomatoid tumour

3 Epithelioid MM Epithelial cells Eosinophilic cytoplasm, bland nuclei Mitoses Infrequent Histologic patterns Papillary, tubulary,microglandular, sheet-like Small cell, clear cell, deciduoid Differential diagnosis Adenocarcinomas and others epithelial tumours

4 Sarcomatoid MM Spindle cells Fascicules or solid sheets Spindle cell or pleomorphic sarcoma Sarcomatoid (pleomorphic) carcinoma of the lung Synovialosarcoma

5 Biphasic MM 30% Both epithelioid and sarcomatoid pattern Each component represent at least 10% of the tumour Percentage will increase with more thorough tumour sampling

6 Desmoplastic MM Dense collagenized tissue separated by atypical cells arranged in a storiform or patternless pattern Differential diagnosis with a benign pleuritis

7 Antibody panel «Mesothelial» markers Calretinin Cytokeratin 5/6 WT-1 HBME-1 Thrombomodulin Mesothelin D2-40 Podoplanin «Epithelial» markers P/mCEA B72.3 Ber-Ep4 CD15 MOC-31 Ordonez N Hum Path 2007; 38:1-16

8 Antibody panel Tissue-associated markers TTF-1 Estrogen and progesterone R P 63 RCC Ma «Mesenchymal» markers Actin Desmin CD34 CD 31 CD 10 bcl2

9 Guidelines for the IHC No individual IHC mesothelial marker that provide 100% specificity and high sensitivity for the diagnosis of MM No epithelial marker that provide high sensitivity and 100% negative predictive value for the diagnosis of EMM No mesenchymal marker that provide high sensitivity and 100% negative predictive value for the diagnosis of SMM

10 Mesothelial positive markers Commonly expressed in mesotheliomas but not in carcinomas and in sarcomas Recently recognized 1. Calretinin 2. Cytokeratin 5/6 3. WT-1 4. HBME-1 5. Thrombomodulin 6. Mesothelin 7. D Podoplanin 9. EMA

11 1. Calretinin Calcium binding protein Variety of tissues Mesothelial cells, adipocytes, neural tissue, sex cord tumours Polyclonal AB Zymed 1:100 Stain : Cytoplasm Combination of both nuclear and cytoplasmic staining more specific for mesothelial differentiation

12 2. Cytokeratin 5/6 Antigen First described in 1985 Blobel JA Am J Path 1985;121: Antibody D5/16B4 XM26 (against keratin 5) Staining Cytoplasmic Focal : false negative result on biopsy

13 3. WT-1 protein One of the most recent mesothelioma marker Amin KM Am J Pathol 1995;146: Antibody 6F-H12 Dako 1:400 Stain Nuclear positivity EM Cytoplasmic positivity SM

14 4.Thrombomodulin First positive mesothelioma marker Collins CL Am J Pathol 1992;141: Surface glycoprotein Variety of epithelial types as well as mesothelium and endothelium Staining of small blood vessels Membranous staining pattern Good sensitivity and low specificity

15 5. Mesothelin 40-kd cell surface glycoprotein of unknown function Antigenic target of the K1 monoclonal antibody 5B2 antimesothelin antibody Ordonez N Mod Pathol 2003;27:192-7 Membranous staining pattern Good sensitivity and low specificity

16 6. HBME-1 Monoclonal antibody directed against the surface of the microvilli of mesothelial cells Precise antigen unknown Staining Membranous in mesotheliomas Cytoplasmic in adenocarcinomas Overlapping staining pattern

17

18 7.D2-40 and 8. Podoplanin Podoplanin is the most recently recognized of the positive MM 38Kd mucin type transmembran protein identified in the surface of rat glomerular epithelial cells - podocytes- Oncofoetal M2A antigen expressed in germ cells Lymphatic endothelial cells, epithelial cells of the choroid plexus, alveolar cells type 1, osteoblast, peritoneal mesothelial cells Antibody: D2-40 Staining: membranous pattern

19 9. EMA Variety of epithelial cells Monoclonal antibody: E29 Staining Membranous in mesothelioma Cytoplasmic in carcinoma No staining in normal and reactive mesothelial cells

20

21 Antibody Epithelial MM % Sarcomatous MM % Adenocarcinoma% Squamous carcinoma% Renal cell carcinoma% Calretinin CytoK HBME WT Negative 0-4 Thrombomoduline Mesothelin D (weak) Podoplanin serous Marchevsky A.M. Arch Pathol Lab Med 2008;132: Ordonez N Hum Path 2007; 38:1-16

22 Positive epithelial markers Commonly expressed in carcinomas but not in mesotheliomas Large number of positive carcinoma markers 1. P/mCEA 2. MOC Ber-Ep4 4. B CD15

23 1. CEA First IHC marker accepted as useful for the distinction betwen EM and ADC mcea Good sensitivity and high specificity pcea High sensitivity and low specificity

24 Glycoprotein markers Ep-CAM The epithelial cell adhesion molecule is a 40-kd transmembrane glycoprotein that is expressed in most epithelial cells A large number of monoclonal antibodies that recognize Ep-CAM are commercially available 2. Ber-EP4 2 glycoprotein 30 and 40 KD from the MCF-7 breast carcinoma cell line Staining: membranous 3. MOC-31 Small cell lung carcinoma cell line (GSL-1) High sensitivity and high specificity

25 4. B72.3 One of the earliest positive carcinoma markers proved to be useful in the diagnosis of MM Monoclonal antibody that react with a tumor associated protein TAG-72 High sensitivity and high specificity

26 5. Leu-M1(CD15) One of the earliest negative mesothelial markers Brown RW Hum Pathol 1993;24: High specificity but low sensibility

27 Tissue-associated markers Restricted expression in carcinomas None expression in mesothelioma 1. TTF-1 2. ER and PR 3. P63 4. RCC Ma

28 1.Thyroid Transcription Factor 1 Nuclear transcription factor Thyroid epithelial cells and type 2 pneumocytes Nuclear TTF-1 expression 85% of lung adenocarcinomas Highest expression in well differentiated tumours Lower expression in poorly differentiated adenocarcinomas and undifferentiated tumors

29 2. Estrogen and progesterone receptors Serous carcinoma of the ovary and the peritoneum ER: 93% ; PR: 29% Ordonez N Hum Pathol 2005;36: Barnetson et al Am J Clin Pathol 2006;125:67-76 Breast adenocarcinoma ER: 72%; PR: 52% ER + PR: 84 % Lee BH et al AM J Clin Pathol 2000; 117: Lack of expression in malignant mesothelioma

30 3. P 63 Nuclear transcription factor Basal cells of the stratified epithelia, prostate, salivary glands and breast and in myoepithelial cells Squamous carcinoma of various primary sites including the lung None or confined expression in mesothelioma

31 4. Renal cell carcinoma marker 200-kd glycoprotein present in the normal proximal tubule of the kidney Monoclonal antibody RCA Ma Conventional renal cell carcinoma Papillary renal cell carcinoma 75%-95% Chromophobe RCC 0%-45% High sensitivity and high specificity

32 Antibody Epithelial MM % Sarcomatous MM % Adenocarcinoma% Squamous carcinoma% Renal cell carcinoma% pcea mcea 3 81 BerEp B CD MOC TTF-1 negative 0 lung 72 other Ne negative Lewis B Application of immunohistochemistry to the diagnosis of malignant mesothelioma Marchevsky A.M. Arch Pathol Lab Med 2008, 132,

33 Cytokeratin subsets Pan keratins AE1/AE3 KL1 CK 5.6 CK7; CK20; CK 8/18

34 Mesenchymal positive markers Commonly expressed in sarcomas but not in mesotheliomas 1. Actine 2. Desmine 3. CD CD CD bcl2

35 Epithelioid MM and adenocarcinoma 2 positive mesothelial markers Calretinin Sensibility 93,8% - Specificity 96,3% Mesopath Cytokeratin 5/6 WT1, podoplanin.. 2 negative epithelial markers MOC-31, Ber-EP4, B72.3, mcea TTF1 may confirm the lung origin of the tumour Estrogen receptor may confirm the breast origin of the tumour or the papillary serous carcinoma of the ovary and peritoneum

36 Epithelioid mesothelioma

37 calretin

38 TTF 1

39 Biphasic mesothelioma

40 Cytokeratin 5.6

41 calretin

42

43 Usefulness of cytokeratin KL1

44

45 Invasion in epithelioid pleural mesothelioma

46 Epithelioid mesothelioma versus squamous carcinoma Positive mesothelial markers Calretinin, CK 5.6 WT1 absent in squamous carcinoma Positive squamous cell carcinoma markers MOC-31, Ber-EP4, CEA, P63 strongly and invariably expressed in Squamous cell carcinoma

47 Epithelioid mesothelioma and renal cell carcinoma Positive mesothelial markers Calretin RCC 0%-10%-EM 82% Podoplanin, CK 5.6 Less sensitive WT-1 RCC 1%- EM 43%- 80% Positive carcinoma markers CD 10 RCC 81%-100% - EM 48%-54% RCC marker RCC 50%-55% - EM 8%- 26% Renal origin of the tumour

48 Atypical mesothelial hyperplasia or superficial mesothelial proliferation of undetermined prognosis

49 IHC for distinction between benign mesothelial reaction and MM Antibody Benign atypical mesothelial proliferation Malignant mesothelioma Keratin AE1/AE EMA +/- +++ P53 +/- +++ Desmin ++ +/- Application of immunohistochemistry to the diagnosis of malignant mesothelioma Marchevsky A.M. Arch Pathol Lab Med 2008, 132,

50 Spindle cell tumours 1. Sarcomatoid mesothelioma 2. Sarcomatoid carcinoma 3. Solitary pleural fibrous tumour 4. Sarcoma

51 1. Sarcomatoid mesothelioma Positive mesothelial markers Calretinin : 5%-88% C 5.6 :13%; WT1:13%; thrombomodulin: 13% Cytokeratin KL1 95% > AE1/AE3 89% > CK5.6 28% Mesopath Positive mesenchymal markers CD 34 : 0% Bcl-2 : 0% Desmin : 0%-5% SMA : 60% Kushitani K. Pathol Intern 2008;58:75-83 KL1

52 MMD : diagnosis Obvious sarcomatoid areas Necrosis without connective tissue Locoregional invasion: lung, mucle, adipose tissue Metastasis : bone Cytokeratin positivity Mesothelial cells in the adipose tissue No usefulness ih the fibrous pleural lesion

53 2. Sarcomatoid carcinoma of the lung Mesothelial markers Calretinin 66,7% Epithelial markers CK 100% - EMA Mesenchymal markers Desmin: 0% SMA: 50% EMA

54 Sarcomatoid carcinoma of the lung There is no useful IH marker for differentiating sarcomatoid mesothelioma from sarcomatoid carcinoma Clinical history, lung tumour

55 3. Solitary pleural fibrous tumour Positive mesothelial markers CK 0% Positive mesenchymal markers CD 34 Bcl2

56 Solitary pleural fibrous tumour CD 34

57 4. Sarcoma Positive epithelial markers Cytokeratin less than 7%; weak and focal EMA : 10% Positive mesothelial markers Calretinin : 30 % WT-1 : 57% Positive mesenchymal markers

58 Sarcoma If tumour cells have diffuse positive results for CK, the tumour is probably mesothelioma If tumour cells are CK negative, the tumour is probably true sarcoma Combination of cytokeratins is the most useful panel for differentiating sarcomatoid mesothelioma from true sarcoma

59 Conclusion IHC is an essential tool for the diagnosis of mesothelioma Current markers Excellent sensibility and specificity for the distinction between MM and metastatic adenocarcinomas Lower accuracy for the distinction between epithelial MM and other epithelial tumours Weak accuracy for the diagnosis of sarcomatous MM Need of consensus or evidence-based guidelines

60 Acknowledgment : French Mesopath Group Galateau-Sallé F Abdalsamad I Begueret H Brambilla E Capron F Coulet A Delajartre A-Y Garbe L Groussard O Piquenot JM Thivolet F Vignaud JM PNSM : Programme National de Surveillance des Mésothéliomes 1998-

61 David Meyronet Samir Benabidallah Lara Chalabreysse Centre de Pathologie Est Hospices Civils de Lyon

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