Workshop: Pathology of Mesothelioma
CONTRIBUTION OF ELECTRON MICROSCOPY T O THE DIAGNOSIS OF PLEURAL MESOTHELIOMA* P. St0ebner.t J. F. Bernaudin,$ M. Nebut,$ and F. Bassets ?Service de Pathologie Cellulaire CHU de Grenoble 38700 La Tronche, France SDkpartement de Recherches sur les Affections Respiratoires et I ‘Environnement Centre Hospitalier Intercommunal 94010 Creteil. France Q Clinique de Pneumo-Phtisiologie Hhpital Laennec et, Hapita1 Bichat Paris, France
The contribution of scanning and electron microscopy to the diagnosis of suspected pleural mesotheliomas has been under investigation. The diagnosic value of the scanning electron microscopy has not been found to be decisive in pleural effusions. By contrast, the transmission electron microscopy was of great value, particularly in lesions or tumors composed mainly of spindle cells. The finding of fibroblastoid mesothelial, or transitional cells, alongside typical mesothelial cells is a clue to the diagnosis of mesotheliomas. The diagnosis of pleural mesothelioma is often difficult to ascertain when using only optical microscopy. Other methods such as histochemistry’ may reduce the diagnosis uncertainty in some cases. However, a t present it seems that electron microscopic study is one of the most reliable approaches, as previously described by two of us.2JoJ1 The purpose of this paper is to report and comment how the scanning and transmission electron microscopy (SEM and TEM) can be helpful for the diagnosis of mesothelioma cases unconfirmed by optical microscopy. This study has been carried out on specimens processed for TEM or for light microscopy. CHARACTERISTICS OF
MALIGNANT MESOTHELIALC E L L S ELECTRONMICROSCOPY (TEM)
IN
TRANSMISSION
Previous ultrastructural studies of 25 malignant pleural mesothelioma cases (2, 9) have shown that these tumors are composed of three main groups of cells: 1. “Epithelial” cells that resembled normal mesothelial cells: they possessed a
polarity with apical junctional systems and/or desmosomes. Their nuclei were irregularly shaped, with a freely dispersed chromatin and a large nucleolus. In the cytoplasm, microfilaments were abundant and forming bundles. Glycogen was usually observed. No secretory granule was visible (FIGURE1). 2. Fusiform cells that looked like atypical fibroblasts, with an irregular nuclear membrane, numerous microfilaments, but without microvilli, basement membrane and juctional systems. *Supported in part by contract INSERM 41-76-73
75 I 0077-8923/79/033&0751 $01.75/0@ 1979. NYAS
752
Annals New York Academy of Sciences
FIGURE I . Tumoral mesothelial cell. g4lycogen. j-Junction. mv-Microvilli. marker line
-
2 1.
3. Transitional (or fibroblastoid mesothelial) cells which had the pattern of fusiform cells with superadded characteristics of epithelial cells such as microvilli, junctional systems and/or hemidesmosomes with associated basement membrane (FIGURE 2).
Stoebner et al.: Electron Microscopy
753
The contribution of the TEM, after usual fixation and embedding for TEM, may be helpful for the diagnosis of atypical mesotheliomas appearing in optical microscopy
like spindle*ell tumors. The discovery of typical mesothelial cells associated to transitional cells allowed us to ascertain the diagnosis of mesotheliomas. In some cases with epithelial pattern in light microscopy the ultrastructural findings allowed to differentiate mesotheliomas from secondary tumors of the pleura. Thus, the presence of transitional cells associated with mesothelial cells is a clue to the diagnosis of mesothelioma. APPRAISAL OF SCANNING ELECTRONMICROSCOPY (SEM) DIAGNOSIS OF PLEURAL MESOTHELIOMAS
I N THE
I . Tissue specimens were examined by SEM in one confirmed mesothelioma case. The pleural surface had various patterns (FIGURE3). Some areas showed finger-like papillary protrusions, others were smooth. The covering cells were loosely imbricated. Numerous red blood cells were observed in widened intercellular spaces. The surface of these cells had microvilli and/or bleb-like membrane projections. In other areas no covering cell was observed. All these findings were confirmed by TEM study. 2. Malignant mesothelial cells were observed in pleural effusions from three other
FIGURE2. Transitional (or fibroblastoid) mesothelial cell. c-Microcavity between two cells. col-Collagen, marker line 5 fi.
-
Annals New York Academy of Sciences
154
FIGURE 3. Surface structure of mesothelioma. a-Finger-like papillary processes, marker line 100 p. CFlat zone, marker line 10 p. c-Mvlesothelial cell surface of pustulous type, marker line I p. d-Spaced microvilli and blebs on an other mesothelial cell, marker line 2 p.
- -
-
-
755
Stoebner et al.: Electron Microscopy
-
FIGURE 4. Surface structure of malignant mesothelial cells. a-Clusters. marker line 10 p. &Isolated cells with blebs. marker line 5 p. c, d-Isolated cells with microvilli and small velamentous projections (*), marker line 1 g.
-
756
Annals New York Academy of Sciences
confirmed mesothelioma cases. The cells were pleiomorphic - some were hyperplastic mesothelial cells, others were modified mesothelial cells resembling those found in noncarcinomatous effusions." The surface pattern of free malignant cells was different, sometimes similar to the pattern observed in the tumor specimen, otherwise it was microvillous with scattered small blebs or thin velamentous projections. The same observations were made in cell clusters (FIGURE 4). 3. Our findings in SEM corroborate the observations made by other^.'*'.^*^*" However, it is still difficult to diagnose mesothelioma by SEM alone. USE OF SEM
AND
TEM FOR RETROSPECTIVE
DIAGNOSIS
Specimens from 13 pleural tumors have been studied retrospectively by SEM and TEM. The tissue samples have been previously fixed in Bouin's or formalin fixatives, and either embedded in paraffin or stored in fixative. Some of these tumors remained unclassified or even excluded by the French Mesothelioma Register Panel. Procedure (FIGURE 5 )
Three 20 pmm thick sections were made from the blocks and settled on slides. One of those was stained in order to choose the significant area. The SEM study was carried out on another section carefully deparaffinized, then osmicated and finally dehydrated. The significant area was selected by comparison with the stained section and cut off with a glass cutter. It was examined after C02critical point drying and coating with gold palladium. The TEM study was carried out on the third section, after embedding which was done by sticking a resin filled gelatin capsule on the chosen area. Tissues stored in fixative were easier to handle. They were sliced, washed, and the procedure used was the usual one for SEM or TEM examination. Results
The following two examples are provided to investigate the possibilities and limitations of the method. The first case (M 219) was a pleural tumor excluded from the French Mesothelioma Register. The optical diagnosis was pachypleuritis. The tissue was fixed in Bouin's solution and paraffin-embedded. SEM and TEM examinations were performed following the above mentioned technique. By SEM, a few cells surrounded by collagen had microvilli. By TEM, characteristic features of fibroblastoid mesothelial (transitional) cells were observed (FIGURE 6). The second case (54 XP 78) was a pleural tumor stored in formalin for about two years. Semi-thin sections were prepared as indicated above. On toluidine blue stained sections, many cavitations, sometimes with a metachromatic content, were seen in the proliferation. By TEM examination, microvilli were seen lining these cavitations. All cells had microfilaments, rough surfaced reticulum and numerous mitochondria. Junctional systems and apical microvilli were observed. The diagnosis of mesothelioma was then ascertained (FIGURE 7).
Stoebner et al.: Electron Microscopy
757
DISCUSSION Presently the retrospective use of SEM was not very helpful for the diagnosis. By contrast, the TEM examination of tissues previously embedded in paraffin or stored in fixative led to conclusive results. This technique has already been used, more or less modified, by several investigators since 19666and has been recently reinvestigated by Johannessen.’ Thus as far as pleural pathology is concerned, this technique appears useful for solving puzzling diagnostic problems. T H I C K
(20m) P A R A F F I N
S E C T I O N S
i D E P A R A F F I N I Z A T I O N Toluene 3 x 10 min. Alcohol Alcohol - Ether
t Os O 4
2 0 min.
D E H Y D R A T I O N Aceton 4 x 10 min. in grading concentrations critical point drying
S E M FIGURE
embedding
T E M
5. Procedure for Retrospective TEM Diagnosis on Paraffin Embedded Material.
The quality of the ultrastructural observations was dependent on the preservation of the recovered tissue. It was excellent with formalin stored specimens, only good with paraffin embedded tissues and poor when fixations other than formalin were used. Despite this fact, it was always possible to differentiate pachypleuritis from purely fibrous mesotheliomas. The characteristic features of the fibroblastoid mesothelial (transitional) cells were always perserved, and only the finding of these cells allowed us to assert the diagnosis of mesothelioma.’ In conclusion, these electron microscopic studies demonstrate that there are
758
Annals New York Academy of Sciences
FIGURE 6. a-Light microscopic aspect of the tumor (case M 219; G x 372). b-Fibroblastoid c-Microfilamentous bundles in a similar cell. d-Microvilli mesothelial cell with microvilli (*) and desmosome (+). (4).
Stoebner et al.: Electron Microscopy
759
FIGURE 7. a-Light microscopic aspect of the tumor (case 54 XP 78; G x 372). b, c and d S h o w characteristic features of the tumor cells, namely microvilli (b). microfilaments (c), and a junctional complex (d).
760
Annals New York Academy of Sciences
characteristic ultrastructural patterns for mesothelioma that ascertain the diagnosis of atypical forms as defined by light microscopy. So, all pleural biopsies from clinically suspected mesotheliomas should be resinembedded. If necessary the ultrastructural study might be done. REFERENCES 1.
2. 3.
4. 5.
6. 7. 8. 9. 10.
I I.
12. 13.
BERLINER. J. A.. M. JANSSEN & C. MCLATCHIE. 1978. The use of scanning electron microscopy in the diagnosis of malignancy in human serous effusions. Scanning Electron Microscopy, SEM Inc. Vol. 11: 797. BERNAUDIN. J. F. 1977. Caractbres ultrastructuraux des cellules mtsothtliales tumorales (Etude de 15 mtsotht-liomes pleuraux malins). Thtse Mtd. Paris, France. DIONNE, G. P. & N. S. WANG.1977. A scanning electron microscopic study of diffuse mesothelioma and some lung carcinomas. Cancer 40:707. DOMAGALA, W. & S. WOYKE.1975. Transmission and scanning electron microscopic studies of cells in effusions. Acta Cytologica 1 9 214. JAGUEUX, M.. R. PIERON & J. M. COSSET.1972. Donntes actuelles sur les mtsothtliomes malins. Caracttrisation chimique de I'acide hyaluronique. J. Fr. M U . Chir. Thorac. 26: 5. LEHNER, T., R. E. N U N N& A. G. E. PEARSE.1966. Electron microscopy of paraffin embedded material in amyloidosis. J. Path. Bact. 91: 297. JOHANNESSEN, J. V. 1977. Use of paraffin material for electron microscopy. Pathol. Annual. 12: 189. KRIVINKOVA, H., J. PONTEN& T. BLONDAL.1976. The diagnosis of cancer from body fluids. Acta path. microbiol. scand. Sec. A, 84: 455. STOEBNER, P., G. MIECH.A. SENGEL & J. P. WITZ. 1970. Notions d'ultrastructure pleurale. I1 Les mtsothtliomes. Press. Med. 78: 1179. 1976. Apport de la microscopie STOEBNER, P., S. AUGUSSEAU & C. MOURIQUAND. tlectronique (balayage et transmission) i I'ttude des liquides pleuraux non ntoplasiques. Arch. Anat. Cytol. Path. 2 4 135. STOEBNER, P.. J. J. ADNET.1978. Mtsothtliomes pleuraux. InttrCt du microscope Clectronique dans leur diagnostic positif et difftrentiel. Biol. Cell. 3 3 50a. SUZUKI. Y.,J. CHURG& M. KANNERSTEIN. 1976. Ultrastructure of human malignant diffuse mesothelioma. Am. J. Pathol. 8 s 241. TAKENAGA, A,, M. MATSUDA, T. HORAI, H. IKEGAMI & S . HATTORI. 1977. Scanning Electron microscopy in the study of lung cancer. New technique of comparative studies on the same lung cancer cells by light microscopy and scanning electron microscopy. Acta Cytol. 21: 90.
CONTRIBUTION OF ELECTRON MICROSCOPY T O THE DIAGNOSIS OF PLEURAL MESOTHELIOMA* P. St0ebner.t J. F. Bernaudin,$ M. Nebut,$ and F. Bassets ?Service de Pathologie Cellulaire CHU de Grenoble 38700 La Tronche, France SDkpartement de Recherches sur les Affections Respiratoires et I ‘Environnement Centre Hospitalier Intercommunal 94010 Creteil. France Q Clinique de Pneumo-Phtisiologie Hhpital Laennec et, Hapita1 Bichat Paris, France
The contribution of scanning and electron microscopy to the diagnosis of suspected pleural mesotheliomas has been under investigation. The diagnosic value of the scanning electron microscopy has not been found to be decisive in pleural effusions. By contrast, the transmission electron microscopy was of great value, particularly in lesions or tumors composed mainly of spindle cells. The finding of fibroblastoid mesothelial, or transitional cells, alongside typical mesothelial cells is a clue to the diagnosis of mesotheliomas. The diagnosis of pleural mesothelioma is often difficult to ascertain when using only optical microscopy. Other methods such as histochemistry’ may reduce the diagnosis uncertainty in some cases. However, a t present it seems that electron microscopic study is one of the most reliable approaches, as previously described by two of us.2JoJ1 The purpose of this paper is to report and comment how the scanning and transmission electron microscopy (SEM and TEM) can be helpful for the diagnosis of mesothelioma cases unconfirmed by optical microscopy. This study has been carried out on specimens processed for TEM or for light microscopy. CHARACTERISTICS OF
MALIGNANT MESOTHELIALC E L L S ELECTRONMICROSCOPY (TEM)
IN
TRANSMISSION
Previous ultrastructural studies of 25 malignant pleural mesothelioma cases (2, 9) have shown that these tumors are composed of three main groups of cells: 1. “Epithelial” cells that resembled normal mesothelial cells: they possessed a
polarity with apical junctional systems and/or desmosomes. Their nuclei were irregularly shaped, with a freely dispersed chromatin and a large nucleolus. In the cytoplasm, microfilaments were abundant and forming bundles. Glycogen was usually observed. No secretory granule was visible (FIGURE1). 2. Fusiform cells that looked like atypical fibroblasts, with an irregular nuclear membrane, numerous microfilaments, but without microvilli, basement membrane and juctional systems. *Supported in part by contract INSERM 41-76-73
75 I 0077-8923/79/033&0751 $01.75/0@ 1979. NYAS
752
Annals New York Academy of Sciences
FIGURE I . Tumoral mesothelial cell. g4lycogen. j-Junction. mv-Microvilli. marker line
-
2 1.
3. Transitional (or fibroblastoid mesothelial) cells which had the pattern of fusiform cells with superadded characteristics of epithelial cells such as microvilli, junctional systems and/or hemidesmosomes with associated basement membrane (FIGURE 2).
Stoebner et al.: Electron Microscopy
753
The contribution of the TEM, after usual fixation and embedding for TEM, may be helpful for the diagnosis of atypical mesotheliomas appearing in optical microscopy
like spindle*ell tumors. The discovery of typical mesothelial cells associated to transitional cells allowed us to ascertain the diagnosis of mesotheliomas. In some cases with epithelial pattern in light microscopy the ultrastructural findings allowed to differentiate mesotheliomas from secondary tumors of the pleura. Thus, the presence of transitional cells associated with mesothelial cells is a clue to the diagnosis of mesothelioma. APPRAISAL OF SCANNING ELECTRONMICROSCOPY (SEM) DIAGNOSIS OF PLEURAL MESOTHELIOMAS
I N THE
I . Tissue specimens were examined by SEM in one confirmed mesothelioma case. The pleural surface had various patterns (FIGURE3). Some areas showed finger-like papillary protrusions, others were smooth. The covering cells were loosely imbricated. Numerous red blood cells were observed in widened intercellular spaces. The surface of these cells had microvilli and/or bleb-like membrane projections. In other areas no covering cell was observed. All these findings were confirmed by TEM study. 2. Malignant mesothelial cells were observed in pleural effusions from three other
FIGURE2. Transitional (or fibroblastoid) mesothelial cell. c-Microcavity between two cells. col-Collagen, marker line 5 fi.
-
Annals New York Academy of Sciences
154
FIGURE 3. Surface structure of mesothelioma. a-Finger-like papillary processes, marker line 100 p. CFlat zone, marker line 10 p. c-Mvlesothelial cell surface of pustulous type, marker line I p. d-Spaced microvilli and blebs on an other mesothelial cell, marker line 2 p.
- -
-
-
755
Stoebner et al.: Electron Microscopy
-
FIGURE 4. Surface structure of malignant mesothelial cells. a-Clusters. marker line 10 p. &Isolated cells with blebs. marker line 5 p. c, d-Isolated cells with microvilli and small velamentous projections (*), marker line 1 g.
-
756
Annals New York Academy of Sciences
confirmed mesothelioma cases. The cells were pleiomorphic - some were hyperplastic mesothelial cells, others were modified mesothelial cells resembling those found in noncarcinomatous effusions." The surface pattern of free malignant cells was different, sometimes similar to the pattern observed in the tumor specimen, otherwise it was microvillous with scattered small blebs or thin velamentous projections. The same observations were made in cell clusters (FIGURE 4). 3. Our findings in SEM corroborate the observations made by other^.'*'.^*^*" However, it is still difficult to diagnose mesothelioma by SEM alone. USE OF SEM
AND
TEM FOR RETROSPECTIVE
DIAGNOSIS
Specimens from 13 pleural tumors have been studied retrospectively by SEM and TEM. The tissue samples have been previously fixed in Bouin's or formalin fixatives, and either embedded in paraffin or stored in fixative. Some of these tumors remained unclassified or even excluded by the French Mesothelioma Register Panel. Procedure (FIGURE 5 )
Three 20 pmm thick sections were made from the blocks and settled on slides. One of those was stained in order to choose the significant area. The SEM study was carried out on another section carefully deparaffinized, then osmicated and finally dehydrated. The significant area was selected by comparison with the stained section and cut off with a glass cutter. It was examined after C02critical point drying and coating with gold palladium. The TEM study was carried out on the third section, after embedding which was done by sticking a resin filled gelatin capsule on the chosen area. Tissues stored in fixative were easier to handle. They were sliced, washed, and the procedure used was the usual one for SEM or TEM examination. Results
The following two examples are provided to investigate the possibilities and limitations of the method. The first case (M 219) was a pleural tumor excluded from the French Mesothelioma Register. The optical diagnosis was pachypleuritis. The tissue was fixed in Bouin's solution and paraffin-embedded. SEM and TEM examinations were performed following the above mentioned technique. By SEM, a few cells surrounded by collagen had microvilli. By TEM, characteristic features of fibroblastoid mesothelial (transitional) cells were observed (FIGURE 6). The second case (54 XP 78) was a pleural tumor stored in formalin for about two years. Semi-thin sections were prepared as indicated above. On toluidine blue stained sections, many cavitations, sometimes with a metachromatic content, were seen in the proliferation. By TEM examination, microvilli were seen lining these cavitations. All cells had microfilaments, rough surfaced reticulum and numerous mitochondria. Junctional systems and apical microvilli were observed. The diagnosis of mesothelioma was then ascertained (FIGURE 7).
Stoebner et al.: Electron Microscopy
757
DISCUSSION Presently the retrospective use of SEM was not very helpful for the diagnosis. By contrast, the TEM examination of tissues previously embedded in paraffin or stored in fixative led to conclusive results. This technique has already been used, more or less modified, by several investigators since 19666and has been recently reinvestigated by Johannessen.’ Thus as far as pleural pathology is concerned, this technique appears useful for solving puzzling diagnostic problems. T H I C K
(20m) P A R A F F I N
S E C T I O N S
i D E P A R A F F I N I Z A T I O N Toluene 3 x 10 min. Alcohol Alcohol - Ether
t Os O 4
2 0 min.
D E H Y D R A T I O N Aceton 4 x 10 min. in grading concentrations critical point drying
S E M FIGURE
embedding
T E M
5. Procedure for Retrospective TEM Diagnosis on Paraffin Embedded Material.
The quality of the ultrastructural observations was dependent on the preservation of the recovered tissue. It was excellent with formalin stored specimens, only good with paraffin embedded tissues and poor when fixations other than formalin were used. Despite this fact, it was always possible to differentiate pachypleuritis from purely fibrous mesotheliomas. The characteristic features of the fibroblastoid mesothelial (transitional) cells were always perserved, and only the finding of these cells allowed us to assert the diagnosis of mesothelioma.’ In conclusion, these electron microscopic studies demonstrate that there are
758
Annals New York Academy of Sciences
FIGURE 6. a-Light microscopic aspect of the tumor (case M 219; G x 372). b-Fibroblastoid c-Microfilamentous bundles in a similar cell. d-Microvilli mesothelial cell with microvilli (*) and desmosome (+). (4).
Stoebner et al.: Electron Microscopy
759
FIGURE 7. a-Light microscopic aspect of the tumor (case 54 XP 78; G x 372). b, c and d S h o w characteristic features of the tumor cells, namely microvilli (b). microfilaments (c), and a junctional complex (d).
760
Annals New York Academy of Sciences
characteristic ultrastructural patterns for mesothelioma that ascertain the diagnosis of atypical forms as defined by light microscopy. So, all pleural biopsies from clinically suspected mesotheliomas should be resinembedded. If necessary the ultrastructural study might be done. REFERENCES 1.
2. 3.
4. 5.
6. 7. 8. 9. 10.
I I.
12. 13.
BERLINER. J. A.. M. JANSSEN & C. MCLATCHIE. 1978. The use of scanning electron microscopy in the diagnosis of malignancy in human serous effusions. Scanning Electron Microscopy, SEM Inc. Vol. 11: 797. BERNAUDIN. J. F. 1977. Caractbres ultrastructuraux des cellules mtsothtliales tumorales (Etude de 15 mtsotht-liomes pleuraux malins). Thtse Mtd. Paris, France. DIONNE, G. P. & N. S. WANG.1977. A scanning electron microscopic study of diffuse mesothelioma and some lung carcinomas. Cancer 40:707. DOMAGALA, W. & S. WOYKE.1975. Transmission and scanning electron microscopic studies of cells in effusions. Acta Cytologica 1 9 214. JAGUEUX, M.. R. PIERON & J. M. COSSET.1972. Donntes actuelles sur les mtsothtliomes malins. Caracttrisation chimique de I'acide hyaluronique. J. Fr. M U . Chir. Thorac. 26: 5. LEHNER, T., R. E. N U N N& A. G. E. PEARSE.1966. Electron microscopy of paraffin embedded material in amyloidosis. J. Path. Bact. 91: 297. JOHANNESSEN, J. V. 1977. Use of paraffin material for electron microscopy. Pathol. Annual. 12: 189. KRIVINKOVA, H., J. PONTEN& T. BLONDAL.1976. The diagnosis of cancer from body fluids. Acta path. microbiol. scand. Sec. A, 84: 455. STOEBNER, P., G. MIECH.A. SENGEL & J. P. WITZ. 1970. Notions d'ultrastructure pleurale. I1 Les mtsothtliomes. Press. Med. 78: 1179. 1976. Apport de la microscopie STOEBNER, P., S. AUGUSSEAU & C. MOURIQUAND. tlectronique (balayage et transmission) i I'ttude des liquides pleuraux non ntoplasiques. Arch. Anat. Cytol. Path. 2 4 135. STOEBNER, P.. J. J. ADNET.1978. Mtsothtliomes pleuraux. InttrCt du microscope Clectronique dans leur diagnostic positif et difftrentiel. Biol. Cell. 3 3 50a. SUZUKI. Y.,J. CHURG& M. KANNERSTEIN. 1976. Ultrastructure of human malignant diffuse mesothelioma. Am. J. Pathol. 8 s 241. TAKENAGA, A,, M. MATSUDA, T. HORAI, H. IKEGAMI & S . HATTORI. 1977. Scanning Electron microscopy in the study of lung cancer. New technique of comparative studies on the same lung cancer cells by light microscopy and scanning electron microscopy. Acta Cytol. 21: 90.
