Department of Pathology, Chiba Cancer Center, 9 Department of Thoracic Surgery, Chiba East Hospital, Chiba, 2

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1 Pathology International 2009; 59: doi: /j x Original Article Malignant pleural mesothelioma: Clinicopathology of 16 extrapleural pneumonectomy patients with special reference to early stage featurespin_ Kenzo Hiroshima, 1 Toshikazu Yusa, 2 Toru Kameya, 3 Ichiro Ito, 3 Kou Kaneko, 4 Chikabumi Kadoyama, 5 Hirohisa Kishi, 6 Yukio Saitoh, 7 Daisuke Ozaki, 8 Makiko Itami, 8 Takekazu Iwata, 9 Akira Iyoda, 10 Toshiaki Kawai, 11 Ichiro Yoshino 12 and Yukio Nakatani 1 Departments of 1 Diagnostic Pathology and 12 Thoracic Surgery, Graduate School of Medicine, Chiba University, 8 Department of Pathology, Chiba Cancer Center, 9 Department of Thoracic Surgery, Chiba East Hospital, Chiba, 2 Department of Thoracic Surgery, Chiba Rosai Hospital, Ichihara, Departments of 6 Pathology and 7 Thoracic Surgery, Narita Red Cross Hospital, Narita, 3 Department of Pathology, Shizuoka Cancer Center, Sunto, Departments of 4 Pathology and 5 Thoracic Surgery, Saitama Red Cross Hospital, Saitama, 11 Department of Pathology, National Defense Medical College, Tokorozawa and 10 Department of Thoracic Surgery, Kitasato University School of Medicine, Sagamihara, Japan The earliest pathological events in the development of malignant pleural mesothelioma (MPM) are not understood. The aim of the present study was to elucidate the early histopathological features of MPM. A total of 16 extrapleural MPM pneumonectomy patients were investigated. Early stage mesothelioma was arbitrarily defined as a tumor 25 mm in thickness regardless of the nodal status or other organ involvement. Eight of these patients (six with epithelioid, two with biphasic) had early stage mesothelioma by this definition. Macroscopically there was no visible tumor, but the parietal and visceral pleura were thickened and there was focal adhesion between them. Microscopically, the mesothelioma lesions were multifocal and discontinuous on the pleura. In extremely early cases of epithelioid mesothelioma, tumor cells were generally arrayed in a single layer, but papillary proliferation was observed elsewhere. In sarcomatoid mesothelioma, mesothelioma cells proliferated, forming multiple small polypoid nodules on the pleura. Epithelial membrane antigen was helpful to distinguish reactive from neoplastic mesothelium, but glucose transporter-1 was not. Mesothelioma cells disseminate diffusely throughout the parietal and visceral pleura and mediastinal fat tissue before becoming visible. Stage Ia mesothelioma (neoplasm limited to the parietal pleura) would not be observed in daily practice. Correspondence: Kenzo Hiroshima, MD, Department of Diagnostic Pathology, Graduate School of Medicine, Chiba University, Inohana, Chuo-ku, Chiba, Chiba , Japan. kenzo@ faculty.chiba-u.jp Received 31 October Accepted for publication 7 April Key words: early stage, epithelial membrane antigen, GLUT-1, immunohistochemistry, International Mesothelioma Interest Group, mesothelioma, mesothelioma in situ, neoplasm staging, pleura, prognosis Malignant mesothelioma is an aggressive tumor of serosal surfaces, such as the pleura, peritoneum, pericardium, and tunica vaginalis. It is thought that the development of mesothelioma is due to exposure to asbestos fibers from industrial and environmental sources, and its incidence is increasing worldwide as a result of widespread exposure to asbestos. 1 Some patients with mesothelioma, however, lack evidence of exposure. Malignant pleural mesothelioma (MPM) predominantly affects men aged years and is associated with a long latency period between exposure and expression of the disease. 2 4 Without treatment, it is associated with a poor median survival, ranging from 4 to 12 months. 2,3 Patients with MPM commonly present with a pleural effusion associated with dyspnea and chest pain at early stages of the disease. 2,3 A delay in establishing a definitive diagnosis reduces the median survival time. Single-modality treatment regimens for MPM including surgery, radiotherapy, or chemotherapy do not prolong the survival of patients. Multimodality regimens involving extrapleural pneumonectomy (EPP) in combination with adjuvant chemotherapy and radiotherapy in selected patients with MPM have demonstrated prolonged survival. 5,6 According to the International Mesothelioma Interest Group (IMIG) staging system, 7 T1a mesothelioma is a tumor limited to the ipsilateral parietal pleura, and there is no

2 538 K. Hiroshima et al. involvement of the visceral pleura. T1b mesothelioma involves the ipsilateral parietal and visceral pleurae. Stage Ia mesothelioma is defined as T1aN0M0, and stage Ib mesothelioma is T1bN0M0. Because patients with MPM, however, are rarely diagnosed during the early stage, the earliest pathological events in the development of mesothelioma and its mechanisms of progression are not understood. Because the natural history of MPM is poorly understood, and the treatment for invasive mesothelioma is often ineffective, definition and identification of the early histopathological changes of mesothelioma are important and may offer clues to the development of effective anti-mesothelioma therapies. The aim of the present study was therefore to elucidate the early histopathological features of mesothelioma and evaluate the validity of the IMIG staging system, analyzing cases in which the entire ipsilateral pleurae and lungs were resected in EPP. MATERIALS AND METHODS Sixteen mesothelioma patients underwent EPP between 1995 and 2008 in the authors institutes. We arbitrarily defined early stage mesothelioma as a pleural tumor 25mm thick regardless of nodal status or other organ involvement; all others were defined as advanced stage mesothelioma. Eight patients were assigned to the early stage according to this criterion. No treatment, such as chemotherapy, radiotherapy, or pleurodesis had been administered before EPP. The surgical materials were fixed in formalin and sections were removed and embedded in paraffin. The number of sections studied per patient ranged from 24 to 51 (average 35). Four micron paraffin sections were cut and processed with HE and EVG staining. The tumors were subdivided into epithelioid, biphasic, or sarcomatoid mesothelioma according to the World Health Organization classification. 8 The pleural elastic lamina was assessed using EVG staining to determine if the tumor penetrated the parietal or visceral pleura. Five layers have been described in visceral pleura: (i) mesothelial layer; (ii) submesothelial layer; (iii) external elastic lamina; (iv) interstitial layer; and (v) internal elastic lamina. 9,10 Parietal pleura are composed of a layer of mesothelial cells and underlying stroma composed of spindle cells, collagen, and elastic tissue. Internal elastic lamina exists under the mesothelial layer, but in some areas it is separated into thinner lamina, or is fragmented and discontinuous. Fibrofatty tissue separates the parietal pleura from the outer elastic lamina that corresponds to the endothoracic fascia. 11,12 Tumor staging was performed using the IMIG staging system. 7 Immunohistochemistry was performed to confirm the diagnosis of malignant mesothelioma. Histofine Simple Stain MAX-PO(M) and MAX-PO(R) (Nichirei, Tokyo, Japan) were used for monoclonal and polyclonal antibodies in immunostaining. The following markers were studied: polyclonal anti-calretinin antibody (prediluted; Zymed, South San Francisco, CA, USA), monoclonal anti-cytokeratin (anti-ck) 5/6 antibody (prediluted; Nichirei), monoclonal anti-d2-40 antibody (prediluted; Nichirei), monoclonal anti-mesothelin antibody (1:40; Novocastra, Newcastle-upon-Tyne, UK), monoclonal anti-epithelial membrane antigen (EMA) antibody (1:800; Dako, Glostrup, Denmark), monoclonal antidesmin antibody (1:200; Dako), polyclonal anti-glucose transporter (anti-glut)-1 antibody (1:200; Dako), monoclonal anti-ck AE1/AE3 antibody (prediluted; Dako), monoclonal anti-cam5.2 antibody (prediluted; Becton Dickinson, Franklin Lakes, NJ, USA), monoclonal anti-cea antibody (1:500; Dako), and monoclonal anti-berep4 antibody (prediluted; Dako). To improve the staining pattern, the tissues were pretreated with microwaves for 15 min in citrate buffer (10 mmol/l ph 6.0) before staining with anti-calretinin antibody, anti-d2-40 antibody, anti-desmin antibody, anti-glut-1 antibody, anti-ck AE1/AE3 antibody, anti-cam5.2 antibody, and anti-cea antibody; or were heated in an autoclave at 121 C for 15 min before staining with anti-ck 5/6 antibody, and anti-mesothelin antibody. The tissues were digested with trypsin (Zymed) for 30 min at 37 C before staining with anti- BerEP4. Catalyzed signal amplification system (Dako) was used for monoclonal anti-wt1 antibody (1:1000; Dako). Suitable positive and negative controls were included. The interval between surgery and death was defined as the overall survival time. Kaplan Meier curves and survival estimates were calculated, 13 and the log rank test was used to test for differences between groups. P < 0.05 was considered statistically significant. Informed consent was obtained from all patients. Patient 3 RESULTS A 66-year-old man presented with shortness of breath in July There was right pleural fluid. Pathological diagnosis on thoracoscopic biopsy was malignant mesothelioma, and EPP was performed in September. There were no abnormal findings in the removed parietal pleura and lung. Microscopically, there were multiple papillary lesions with capillaries on the parietal pleura (Fig. 1a,b). The largest lesion was 1 mm in diameter and contained larger capillaries, and mesothelioma cells proliferated both on the surface and inside the lesion (Fig. 1c). Mesothelioma cells invaded fat tissue of the parietal pleura. Mesothelioma cells were positive for EMA. Flat, apparently uninvolved portions of the pleural surfaces were covered with reactive mesothelial cells or atypical mesothelial cells. EMA was positive on the cytoplasmic membrane of

3 Malignant mesothelioma at early stage 539 a b c d e Figure 1 Patient 3. Extremely early stage mesothelioma. Macroscopically, there are no nodules in the parietal pleura, visceral pleura, or lung parenchyma. Microscopic findings. (a) Papillary proliferation with capillaries on the parietal pleura. (b) Both papillary and monolayer growth of mesothelioma cells are stained with epithelial membrane antigen (EMA), but reactive mesothelial cells are not. (c) The largest lesion contains larger capillaries, and mesothelioma cells proliferated on and within this lesion. (d) Visceral mesothelial cells are lacking due to acute pleuritis, but glandular structures composed of atypical cells (arrows) are observed in some areas. (e) EMA is positive on the membrane of the atypical cells. This lesion is diagnosed as early invasion into visceral pleura. the atypical mesothelial cells arrayed in a single layer (Fig. 1b). Glandular structures composed of atypical cells were observed in some areas of the visceral pleura (Fig. 1d). EMA was positive on the cytoplasmic membrane of the atypical cells (Fig. 1e). We diagnosed this lesion as early invasion into submesothelial layer of the visceral pleura. There was metastasis to the lymph apparatus in mediastinal fat tissue. This patient was diagnosed as having epithelioid mesothelioma. Because mediastinal fat tissue was invaded, and there was seeding along the wound in the chest wall on thoracoscopy, the tumor was classified as T3N0M0, stage III. The course after the operation was uneventful, but an abdominal mass has developed and recurrence of the tumor is suspected.

4 540 K. Hiroshima et al. a a b b Figure 2 Patient 4. Microscopic findings of early stage epithelioid mesothelioma. (a) Papillary proliferation on the surface of the diaphragm is seen, but there is no invasion into the submesothelial fat tissue. (b) The mesothelioma cells proliferate in a papillary pattern on the surface of visceral pleura, but there is no invasion into the lung. Figure 3 Patient 8. Microscopic findings of early stage sarcomatoid mesothelioma. (a) Multiple solitary nodules composed of spindle cells in the submesothelial tissue (video-assisted thoracoscopic biopsy). (b) The visceral pleura are covered with sarcomatoid lesions (extrapleural pneumonectomy). Patient 4 A 61-year-old man, previously exposed to asbestos, presented with a right pleural effusion discovered when he undertook cholecystectomy for cholecystolithiasis in May Pathological diagnosis of thoracoscopic biopsy suggested malignant mesothelioma, and EPP was performed in September. There was pleural plaque, but no visible nodule in the pleural cavity. Microscopically, mesothelioma cells proliferated in a papillary pattern on the surface of the parietal pleura but did not invade the subpleural fat tissue (Fig. 2a). They proliferated on the surface of the visceral pleura but there was no invasion into the lung (Fig. 2b). This patient was diagnosed as having epithelioid mesothelioma. Because tumor had seeded along the wound in the chest wall on thoracoscopy, the tumor was classified as T3N0M0, stage III. Figure 4 Patient 6. Microscopic findings of early stage epithelioid mesothelioma. Mesothelioma cells embedded in a layer of fibrin coated the uninvolved visceral pleural surface.

5 Malignant mesothelioma at early stage 541 Table 1 Malignant pleural mesothelioma patients treated with extrapleural pneumonectomy Patient no. Histology Sex Age (years) Asbestos exposure Hyaluronic acid (ng/ml) IMIG stage Prognosis (months) Outcome 1 Epithelioid M 62 Present Ib 89 Recurrence 2 Biphasic M 48 Present III 16 Dead 3 Epithelioid M 66 Unknown III 36 Recurrence 4 Epithelioid M 61 Present III 28 Dead 5 Epithelioid M 53 Present ND II 17 Alive 6 Epithelioid M 56 Present III 17 Dead 7 Epithelioid M 68 Present Ib 3 Alive 8 Biphasic M 72 Present III 2 Alive 9 Epithelioid M 40 Unknown III Unknown 10 Epithelioid M 53 Absent II 18 Dead 11 Biphasic M 58 Absent IV 6 Dead 12 Epithelioid M 64 Absent ND III 14 Dead 13 Sarcomatoid M 48 Present III 32 Dead 14 Sarcomatoid M 51 Present II 10 Dead 15 Sarcomatoid M 59 Absent ND II 11 Dead 16 Epithelioid M 44 Unknown III 14 Dead Early stage mesothelioma: mesothelioma without grossly visible tumor. ND, not done; IMIG, International Mesothelioma Interest Group. There appeared local recurrence at the site of drainage after EPP in March 2006, and chemotherapy was performed. The patient died in January Autopsy was not performed. Patient 8 A 72-year-old man, previously exposed to asbestos, presented with a complaint of dry cough in December There was right pleural fluid. Pathological diagnosis of thoracoscopic biopsy was sarcomatoid mesothelioma (Fig. 3a), and EPP was performed in January There was pleural thickening both in the parietal and visceral pleura, but the degree was higher in parietal pleura. There was adhesion between parietal and visceral pleura on upper lobe and in the mediastinal side. Interlobar pleura were also thickened. Microscopically, the tumor cells were spindle-shaped and proliferated in patternless pattern. Expansile nodules pushing borders were observed. Papillary or solid proliferation of epithelial component was also observed, but the sarcomatoid component represented 90% of the tumor. The mesothelioma lesions were discontinuous on the visceral pleura (Fig. 3b). The diagnosis was biphasic mesothelioma. Because tumor had seeded along the wound in the chest wall on thoracoscopy, the tumor was classified as T3N0M0, stage III. Summary of Clinicopathology Hemithoracic radiotherapy was performed after EPP, and the patient was alive without recurrence in February Clinical data for all MPM patients who underwent EPP are shown in Table 1. In Table 2 the size and location of the tumors in early stage mesothelioma are summarized. The early stage mesotheliomas in the present study included two stage Ib, one stage II and five stage III. Six were well differentiated epithelioid mesotheliomas and two were biphasic mesotheliomas. Macroscopically, there was no visible tumor, but the parietal pleura were thickened and there was focal adhesion between parietal and visceral pleura in early stage mesothelioma. Microscopically, the mesothelioma lesions were small and discontinuous, and the pleura adjacent to the lesion were covered with normal mesothelial cells. In extremely early cases (patients 2 4), tumor cells were generally arrayed in a single layer, but papillary proliferation was observed elsewhere. Mesothelioma cells embedded in a layer of fibrin coated the uninvolved visceral pleural surface (Fig. 4). Parietal and visceral pleura were adhered to each other and mesothelioma cells proliferated between them. The thickened pleura were composed of proliferation of mesothelioma cells. The thickness of the pleural tumor was 1 5 mm. We evaluated the immunohistochemical staining pattern of EMA, desmin, and GLUT-1, which are claimed to help in discriminating benign from malignant mesothelial cells in early stage mesothelioma. Immunoreactivity for these antibodies was compared between the areas of reactive mesothelial hyperplasia and apparently malignant mesothelial proliferation concurrently observed in each case (Table 3). EMA was expressed in all mesotheliomas, but not expressed in reactive mesothelium except in one. Desmin was expressed in reactive mesothelium in two cases. GLUT-1 was expressed in two early stage mesotheliomas. Hyperthermic intrathoracic chemotherapy after EPP was performed for six patients. Systemic chemotherapy was

6 542 K. Hiroshima et al. Table 2 Summary of early stage mesothelioma Thickness of the largest lesion (mm) Largest lesion Macroscopic findings Microscopic findings Parietal pleura Visceral pleura Parietal pleural invasion Visceral pleural invasion Other invasion Patient no. 1 Small nodules Small nodules Subpleural fat External elastic lamina Parietal pleura 4 Surface Seeding Mediastinal pleura 4 A few small nodules Subpleural fat (mediastinal pleura) 2 Small nodules in mediastinal pleura 3 None None Subpleural fat Minimal Seeding, mediastinal fat Parietal pleura 1 4 Irregularity on diaphragm Focal thickening Subpleural fat (diaphragm) Surface Seeding Diaphragm 2 5 Thickening Focal thickening Subpleural fat Lung parenchyma Parietal pleura 4 6 Thickening Small nodules Subpleural fat, Lung parenchyma Mediastinal lymph node Mediastinal pleura 5 Diaphragmatic muscle 7 Thickening Focal thickening Subpleural fat Surface Parietal pleura 4 8 Thickening Focal thickening Subpleural fat External elastic lamina Seeding Parietal pleura 5 Table 3 Immunoreactivity in early stage mesothelioma n EMA (%) Desmin (%) GLUT-1 (%) Mesothelioma 8 8 (100) 1 (13) 2 (25) Reactive mesothelium 8 1 (13) 2 (25) 0 (0) EMA, epithelial membrane antigen; GLUT-1, glucose transporter 1. performed for five patients and hemithoracic radiation in one, and radiotherapy at the site of local recurrence in five patients. Five patients were alive at the time of writing. The eight patients with early stage mesothelioma in the present study had 1, 2, and 3 year survival rates of 100%, 67%, and 44%, respectively. The eight patients with advanced stage mesothelioma treated with EPP in the authors institutes had 1, 2, and 3 year survival rates of 57%, 14%, and 0%, respectively (P = ). DISCUSSION The IMIG proposed that the TNM descriptions should be based on surgical and pathological findings, 7 buts there are no guidelines defining the various stages of early invasion. Invasion only to submesothelial tissue cannot be reliably diagnosed as malignant, because histological distinction between reactive mesothelial hyperplasia and mesothelioma is difficult in small biopsy samples. Epithelial mesothelial growth that reaches the subpleural fat tissue is evidence for malignancy. 14 According to International Union Against Cancer TNM classification, which is based on the recommendations of the IMIG TNM staging system, tumor reaching the subpleural fat tissue of the diaphragm is T1 and tumor invading the diaphragmatic muscle is T2. 7 Tumor reaching the subpleural fat tissue under the parietal pleura of the chest wall is T1 and tumor invading the endothoracic fascia is T3 (Fig. 5a). 7 It is T2 if the tumor invades lung parenchyma. 7 Tumors that invade the interstitial layer between external and internal elastic lamina in the visceral pleura are T1 (Fig. 5b). Internal elastic lamina is continuous with the pulmonary interstitium of the interlobular septum. We often observe that mesothelioma penetrates the external elastic lamina and proliferates in the interstitial tissue between two laminas in early stage mesothelioma. Distinguishing visceral from parietal pleural involvement cannot be done with the resolution of current CT and magnetic resonance imaging. 15 It is difficult to determine whether T1a, T1b (or even T2) disease is present if the tumors are staged non-invasively. 7,16 A recent report evaluating integrated CT positron emission tomography and pathological staging after EPP, did not subdivide T1 into T1a and T1b. 17 There are some reports of stage T1a mesotheliomas in therapeutic studies of MPM, but the stage was diagnosed on thoracoscopy and not confirmed with surgical samples on

7 Malignant mesothelioma at early stage 543 Figure 5 Schematic diagram of progression of early stage mesothelioma. (a) Parietal pleura. Tumor is T1 provided it is confined to subpleural fat tissue of chest wall. It is T3 if it invades the endothoracic fascia, and T2 if it invades diaphragmatic muscle. (b) Visceral pleura. Tumor is T1 as long as it does not invade internal elastic lamina in the visceral pleura. It is T2 if it invades lung parenchyma. EPP. 18,19 No instances of T1a MPM based on surgical and pathological staging were identified in patients who underwent thoracotomy for possible resection of MPM. 20,21 The IMIG TNM staging system was published in 1995, but there are no published reports of pathological T1a mesothelioma. Because T1a mesothelioma is thought to progress to T1b immediately during the subclinical stage, separation of T1 into T1a or T1b is not practical. In a report of early stage mesothelioma by Whitaker et al., several microscopic white foci no more than 2 mm in diameter were found on both visceral and parietal pleural surfaces at necropsy in early stage mesothelioma, and no gross pleural tumor was evident. 22 After extensive thoracoscopic experience, Boutin et al. reported that non-specific inflammatory lesions, pachypleuritis, nodules <5 mm, and a combination of pachypleuritis and small nodules were observed in biopsy samples of parietal pleura in stage IA mesothelioma, and the visceral pleura were invaded later. 23 In contrast to their report, the case reported by Whitaker et al. and the present one showed that mesothelioma cells proliferated both on parietal and visceral surface multifocally before the small nodules were observed on thoracoscopy. Early signs of tumor formation in experimentally induced mesothelioma in rodents included numerous small irregularly scattered nodules. 24 Occurrence of multiple lesions on the pleural surface may be due to the dissemination of mesothelioma cells through the pleural cavity. The finding that mesothelioma cells embedded in a layer of fibrin coated the uninvolved visceral pleura (Fig. 4) supports this hypothesis. Alternatively, mesothelioma cells invading submesothelial connective tissue may proliferate quickly and travel to distant sites. In patient 2 the mesothelioma cells were observed in lymphatic vessels, and in patient 3 involvement of the lymph apparatus in mediastinal fat tissue was observed when proliferation of mesothelioma cells on the parietal pleura was scant. From these findings, we propose steps in the progression of early stage mesothelioma before the tumor becomes visible. In epithelioid mesothelioma it develops on parietal pleura as mesothelioma in situ and invades fat tissue. Papillary proliferation occurs on the surface of the pleura, which then grows. In visceral pleura, mesothelioma cells invade the submesothelial layer and external elastic lamina. Mesothelioma cells might invade from the surface of the visceral pleura without leaving mesothelioma lesion on the surface of the pleura. Some area of the surface of the visceral pleura, however, is covered with mesothelioma in a papillary pattern. The lesions in the parietal and visceral pleura are multiple and discontinuous at first. There appear fibrinous exudates on the surface of parietal and visceral pleura, and adhesion of both pleura occurs. In the early stage the adhesion occurs after the absorption of exudates and there may be no neoplastic cells between parietal and visceral pleura. In the advanced stage, however, adhesion occurs after the conglomeration of parietal and visceral tumors, and the nodules become even larger and more confluent (Fig. 6). In sarcomatoid mesothelioma, multiple small polypoid nodules appear on the surface of the parietal pleura, and they fuse together. In visceral pleura, small nodules expand in the pleura invading the submesothelial layer and external elastic lamina, and then they fuse together. Adhesion of parietal and visceral pleura occurs later (Fig. 7). Desmin and EMA are the most useful markers in distinguishing benign from malignant mesothelial proliferations. EMA is preferentially expressed in neoplastic mesothelium, and desmin is preferentially expressed in reactive mesothelium. 25 GLUT-1 is expressed in mesothelioma, but not in reactive mesothelium. 26 In the present study, EMA was expressed in all early stage mesothelioma, but it was expressed only in one case in reactive mesothelium concurrently observed in early stage mesothelioma. The frequency of expression of desmin in reactive mesothelium was not as high as previously reported. 25 GLUT-1 was expressed in two early stage mesotheliomas, and it would not be a sensitive immunohistochemical marker for the diagnosis of early stage mesothelioma. Because treatment for invasive mesothelioma is ineffective, early detection of malignant mesothelioma is important.

8 544 K. Hiroshima et al. Figure 6 Schematic diagram of progression of early stage epithelioid mesothelioma before it becomes visible. Mesothelioma first develops on parietal pleura and invades fat tissue soon after. There is a papillary proliferation on the surface of the pleura. In visceral pleura, mesothelioma cells invade pleura at an early stage, and proliferate inside the pleura. The surface may consist of benign mesothelial cells but in some areas the surface is covered with mesothelioma cells that proliferate in a papillary pattern. Adhesion of parietal and visceral lesions occurs and the nodules become large and confluent. Pleural effusion was the only radiographically abnormal finding, and no nodules were detected on radiography in these patients. Cytology of the aspirated pleural fluid is a logical first step, but it is not conclusive because there is overlap between the cytological atypia of mesothelial hyperplasia and mesothelioma. Furthermore, there is no specific immunohistochemical test or other marker that discriminates between hyperplastic and neoplastic mesothelium. 27 Pathological diagnosis of mesothelioma using small biopsies is also as difficult as cytology. Although papillary architecture and necrosis of mesothelial cells favor mesothelioma in the pleura, invasion is the most decisive indicator of mesothelioma. 14,27 Because MPM often appears on the mediastinal pleura or costophrenic sulcus, video-assisted thoracoscopic surgery offers the distinct advantages over percutaneous pleural biopsy. Clinicians must obtain large specimens including fat tissue beneath the parietal pleura on thoracoscopic biopsy to render accurate pathological diagnosis of the disease. Surgery alone for MPM is associated with a high recurrence rate even if complete resection is achieved. Sugarbaker et al. reported the results of 183 MPM patients who underwent EPP followed by adjuvant chemotherapy and radiotherapy. Survival was 38% at 2 years and 15% at 5 years (median 19 months). 6 Rusch et al. conducted a phase II trial of high-dose hemithoracic radiation after EPP and found that the median survival was 33.8 months for stage I and II tumors but only 10 months for stage III and IV tumors. 28 Recently, Flores et al. analyzed 663 patients with MPM who underwent EPP or pleurectomy/decortications and reported that the overall survival at 5 years for all patients was 12%, and the median survival was 38 months for stage I, 19 months for stage II, 11 months for stage III, and 7 months for stage IV tumors. 29 The prognosis for patients with early stage MPM who underwent EPP was good compared with that for advanced stage MPM in the present study. These results show that EPP with adjuvant therapy prolongs the survival of patients with early stage MPM. ACKNOWLEDGMENTS Figure 7 Schematic diagram of progression of early stage sarcomatoid mesothelioma before it becomes visible. Dome-shaped nodules appear on parietal pleura, and progress to become larger nodules. Spindle-shaped cells proliferate, forming nodules in the visceral pleura, pushing the inner elastic layer down into lung parenchyma, and progress to become confluent. Both parietal and visceral pleura fuse to form thickened pleura. The authors thank Ms Ayaka Sato, Ms Tamiyo Taniguchi, Ms Yoko Hata, and Ms Kazuko Abe for their technical assistance. This study was supported in part by a Grant-in-Aid for Scientific Research (C) of the Japanese Ministry of Education, Culture, Sports, Science, and Technology. REFERENCES 1 Robinson BW, Lake RA. Advances in malignant mesothelioma. N Engl J Med 2005; 353: Hillerdal G. Malignant mesothelioma 1982: Review of 4710 published cases. Br J Dis Chest 1983; 77: Ruffie P, Feld R, Minkin S et al. Diffuse malignant mesothelioma of the pleura in Ontario and Quebec: A retrospective study of 332 patients. J Clin Oncol 1989; 7: Neumann V, Gunthe S, Mulle KM, Fischer M. Malignant mesothelioma: German mesothelioma register Int Arch Occup Environ Health 2001; 74: Sugarbaker DJ, Mentzer SJ, DeCamp M, Lynch TJ Jr, Strauss GM. Extrapleural pneumonectomy in the setting of a multimodality approach to malignant mesothelioma. Chest 1993; 103: 377S 381S. 6 Sugarbaker DJ, Flores RM, Jaklitsch MT et al. Resection margins, extrapleural nodal status, and cell type determine postoperative long-term survival in trimodality therapy of malignant pleural mesothelioma: Results in 183 patients. J Thorac Cardiovasc Surg 1999; 117: 54 63, discussion 63 5.

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