Postgraduate Course 10 Malignant pleural effusion management

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1 ERS International Congress Amsterdam September 2015 Postgraduate Course 10 Malignant pleural effusion management Thank you for viewing this document. We would like to remind you that this material is the property of the author. It is provided to you by the ERS for your personal use only, as submitted by the author by the author Saturday, 26 September :30 13:00 Room 13.2 RAI

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3 Postgraduate Course 10 Malignant pleural effusion management AIMS: To understand the new roles that molecular biology and histo-/cytopathology play in the diagnosis and management (treatment and prognosis) of malignant pleural effusions, and how to use these techniques (processing and data interpretation) in clinical practice. TARGET AUDIENCE: Trainees, pulmonary physicians, thoracic surgeons, pathologists, molecular biologists, and scientists. CHAIRS: P. Astoul (Marseille, France), M. Skrzypski (Sopot, Poland) COURSE PROGRAMME PAGE 09:30 Which material in which disease? 5 S. Fernandez Bussy (Santiago, Chile) 10:15 How to send your material to pathology 82 P. Schnabel (Homburg/Saar, Germany) 11:00 Break 11:30 Interpretation of the results in the clinic 83 A. Scherpereel (Lille, France) 12:15 Atypical mesothelial hyperplasia and other pitfalls in MPE diagnosis 102 F. Galateau-Salle (Caen, France) Faculty disclosures 108 Faculty contact information 109

4 Regular physical activity improves quality of life and fitness in healthy individuals and people living with lung conditions. In partnership with: Farbe/colour: PANTONE 288 CV ERS/ELF ER Journal 210x280 ad_v2 AW.indd 4 29/07/ :23

5 Malignant Pleural Effusion Dr Sebastian Fernandez Bussy Clinica Alemana-Universidad del Desarrollo Interventional Pulmonology Av Vitacura 5951 Santiago, Región Metropolitana CHILE SUMMARY Metastatic disease to the pleura occur much more frequently than primary pleural neoplasms, causing approximately pleural effusions in the USA each year, as compared to Diffuse Malignant Mesothelioma (DMM), which causes approximately 1500 pleural effusions each year. This is a ratio of over 130 pleural metastases for each primary pleural DMM. Metastases to the pleura exhibit characteristics, including pleural effusions, pleural thickening and pleural-based masses, found with primary pleural neoplasms and reactive pleural processes. Common metastases to the pleura include lymphoma, lung cancer and breast cancer; however, malignancies from any primary may metastasize to the pleura. The pleura can be invaded directly from neighboring structures (lung, chest wall -- including breast in some cases -- diaphragm and mediastinum) but most of the pleural malignancies arise, according to necropsy studies, from tumor emboli to the visceral pleura, with secondary seeding to the parietal pleura. The effusion can develop as a direct consequence of neoplastic pleural involvement, but it can also occur in cases with lymphatic blockade at the mediastinal level. Diagnostic thoracentesis should be performed in every pleural effusion not clearly associated to heart failure, hepathic hydrothorax or chronic renal failure, and also in patients with the above conditions not responding to the appropriate treatment. Malignant transudative effusions can occur in lung cancer with obstruction of the mainstem bronchus, but also in lymphoma and other malignancies. Blockade of lymphatic drainage may be invoked as one of the possible pathogenetic mechanisms, but an underlying cardiac failure or other conditions that are usually associated with transudative effusions should be taken into account also. Many studies have shown a large variation in the diagnostic sensitivity of pleural fluid cytological analysis, ranging from 40 87%2. In particular, a cytomorphologic distinction between reactive mesothelial cells, mesothelioma, and metastatic adenocarcinoma, as well as between lymphomas and reactive lymphocytosis, can often be difficult because of significant overlapping cytologic features. Therefore, other procedures, such as immunohistochemistry (IHC) using monoclonal antibodies against tumor markers and chromosomal analysis, complement cytology in the diagnosis of MPEs. IHC staining can be performed on conventional cytology specimens and cell blocks. There have been many reports on the application of different IHC markers in pleural effusion samples to diagnosis MPE and to identify the primary site of origin. Differential cell counts in pleural fluid are never diagnostic, but can help (more than two-thirds of lymphocytic pleural effusions are the result of malignancy or tuberculosis). Pleural fluid eosinophilia (> 10% eosinophils in the total nucleated cell count) is virtually never diagnostic because it can be due to idiopathic (25%), malignant (20%), infectious (20%) or miscellaneous 5

6 benign conditions (35%) [48]. It should be stressed that the higher the percentage of pleural eosinophils (e.g., > 40%), the lower the likelihood of malignancy and the greater the likelihood of an unknown etiology. A negative Adenosin deaminase (ADA) is useful to rule out tuberculous pleuritis in lymphocytepredominant effusions [12]. In addition, extremely high ADA (> 250 U/L) in pleural fluid should raise suspicion of a non-tuberculous effusion, particularly lymphoma or complicated parapneumonic effusion. The yield from sending more than two separate pleural fluid specimens for cytology is very low and should be discouraged: In a study of 603 patients with MPEs, cytological analysis had an overall sensitivity of 50% (95% confidence interval (CI): %) for the first specimen and 57% (95% CI: %) and 58.2% (95% CI: %) for the second and third separate examinations, respectively. For both direct smear/cytospin and cell block preparations, 150 ml or more were recommended, whereas 60 ml were adequate when only direct smear/cytospin was used for the diagnosis of MPE. To separate epithelial mesothelioma from adenocarcinoma, at least two mesothelial (e.g., calretinin, keratin 5/6, WT-1 protein) and two carcinoma (e.g., thyroid transcription factor-1- TTF-1-, CEA, B72.3) markers should be used [56]. In a MPE of unknown origin, a positive TTF- 1 stain strongly suggests a primary non-small-cell lung cancer. Immunocytochemistry in pleural fluid can also help in some therapeutic decisions, like selection of targeted therapy in lung cancer [59], and hormonal therapy. Thus, expression of oestrogen/progestogen receptors or Her- 2-neu may have both diagnostic (breast cancer) and therapeutic implications. Flow cytometry has the advantage of fast and observer independent results and can be very helpful in identifying pleural involvement of hematologic malignancies, and in distinguishing malignant from atypical mesothelial cells. There are an increasing number of MPE patients as the incidences of lung cancer and other malignant cancers have increased. Although cytological or histopathologic changes act as the gold standard of MPE diagnosis, these changes are not observed in the bodies of all patients with MPE. Moreover, many patients are unwilling to undergo a biopsy examination. Many researchers have evaluated multiple assays to find out a necessary to find a highly specific and sensitive MPE marker to improve detection accuracy, reduce patient morbidity and inconvenience, and decrease follow-up financial pressure. A panel of PF tumor markers, including CEA, CA 125, CA 15-3 and CYFRA 21-1, have been tested. Although the measure of these molecular markers can serve as a diagnostic guide, they cannot be used exclusively to confirm a diagnosis. Mesothelin levels have been examined in many series of serum samples; however problems of confounding by kidney function and low sensitivity in presymptomatic individuals have limited its acceptance as a diagnostic marker. In comparison there have been far fewer studies of the role of mesothelin measurements in pleural effusions in a diagnostic setting. Tumor marker measurements in effusions have the potential to be more sensitive than serum measures because proximity of the fluid to the tumor allows continuous sampling of the tumor and because effusions are derived from an already symptomatic group. Mesothelin levels greater than 20 nm in effusions are highly suggestive of malignancy, particularly of MM. With a sensitivity of 67% and specificity of 95% for distinguishing Malignant Mesothelioma (MM) from all other effusions this study confirms that measurement of mesothelin level contributes to the diagnostic 6

7 investigation of patients with pleural effusions. As 33% of MM cases do not have an elevated mesothelin level this test is not sufficient for diagnostic purposes as a stand-alone test. The finding that non-mm mesothelin positive cases are primarily due to other malignancies supports the notion that the value of this test is in differentiating malignant from nonmalignant effusions. One of the early biomarkers, proposed over 30 years ago, for mesothelioma was hyaluronic acid. However, the technically demanding nature of the hyaluronic acid assay limited follow-up studies. Recently, two independent studies have been reported that find combining hyaluronic acid with mesothelin or MPF improves sensitivity for mesothelioma, while maintaining specificity when measured in effusion supernatants. Sensitivity of mesothelin for mesothelioma in a dichotomous differential diagnostic setting has been improved by using additional exclusion markers, such as carcinoembryonic antigen, has been reported. Survivin has been confirmed as a novel member of the inhibitor of apoptosis protein (IAP) family. Reportedly, survivin exerts antiapoptosis effects and regulates cell division in tumors and normal tissues. During tumorigenesis, surviving expression is inversely correlated with apoptosis inhibition but positively correlated with proliferation and angiogenesis. Survivin has been widely studied as a prognostic marker in NSCLC, and it plays a crucial role in regulating cell proliferation, apoptosis, and angiogenesis. Many studies have shown that the positive expression of survivin is significantly related to lymph node metastasis and cancers of late clinical stage, such as MPE. In recent years, surviving has generated much interest as a novel biomarker for MPE diagnosis, and it appears to be a useful marker of MPE. It has been reported that levels of plasma (not serum) fibulin-3 can distinguish healthy persons with exposure to asbestos from patients with mesothelioma, and can further differentiate mesothelioma effusions from other malignant and benign effusions. Although, there is still not enough information about the routine use of fibulin-3. For tissue sample, needle pleural biopsy (US or CT guided) or thoracoscopy, can be used. Pleural malignant mesothelioma (MM) is an aggressive asbestos-induced malignancy. The diagnosis of MM is often difficult and may take several weeks to months to establish. Current guidelines recommend that a diagnosis be based on demonstration of invasion by tumor cells; however, not all patients are able to undergo invasive procedures to obtain a biopsy, and even when tissue is obtained diagnosismay still be difficult. In MM, patients often present with a pleural effusion which can be used for diagnostic purposes. Although there is controversy regarding the cytological analysis of cells from pleural effusions, with sensitivities reported in several studies to be around 30%, the limitation of the technique relates primarily to difficulty in distinguishing MM from benign mesothelial cells. However, a high sensitivity and specificity can be achieved if a standardized approach to cytodiagnosis is used. MPM grows in the pleural cavity diffusely more often than as a localized mass. According to WHO histological classification of tumors of the pleura, diffuse MM is classified into four types; epithelioid, sarcomatoid, desmoplastic, and biphasic mesothelioma. Epithelioid mesothelioma (MPM of epithelial type) is most common, and better prognosis has been reported for patients with MPM of epithelial type than those with MPM of other types. An extrapleural pneumonectomy (EPP), considered to be a curative intent surgery for MPM, is characterized by stripping of the affected parietal pleura from the endothoracic fascia of the chest wall and en bloc removal of the affected parietal pleura and lung. To establish radical cure of MPM, it is therefore indispensable to find earlystage MPM of epithelial type, in which mesothelial proliferation is localized on the serosal surface of parietal pleura. Morphological discrimination between MPM 7

8 of epithelial type and reactive mesothelial hyperplasia (RMH) is difficult, and the most reliable pathological criterion for malignancy is mesothelial proliferation invading deeply into subpleural adipose tissues. When a patient presents with an undiagnosed pleural effusion, thoracentesis is generally the first intervention performed. If no definite cancer cells are apparent in the fluid and no alternative cause for the effusion can be found then thoracoscopy with biopsy will be considered if malignancy is a significant possibility. However, in some patients with significant underlying comorbidities and/or frailty in whom thoracoscopy is considered too high risk, or where thoracoscopy is not available, or declined by the patient, measurement of serum and pleural effusion mesothelin levels is a useful step a positive result dramatically increases the likelihood of there being a pleural malignancy present. Although soluble mesothelin has a useful clinical role in diagnosis because of its high specificity, its sensitivity remains limited. That means that a positive result is informative, but a negative one is not. A better biomarker for pleural malignant mesothelioma has yet to be identified. REFERENCES 1. Lamia H. Shabaan et al. Value of thoracoscopic pleural brush in the diagnosis of exudative pleural effusion. Egyptian Journal of Chest Diseases and Tuberculosis (2012) 61, Georgia Karpathiou et al. Pleural neoplastic pathology. Respiratory Medicine (2015) xx, 1e13 3. Henderson DW, et al. Challenges and controversies in the diagnosis of mesothelioma: Part 1. Cytology-only diagnosis, biopsies, immunohistochemistry, discrimination between mesothelioma and reactive mesothelial hyperplasia, and biomarkers. J Clin Pathol 2013;66: Henderson DW, et al. Challenges and controversies in the diagnosis of malignant mesothelioma: Part 2. Malignant mesothelioma subtypes, pleural synovial sarcoma, molecular and prognostic aspects of mesothelioma, BAP1, aquaporin-1 and microrna. J Clin Pathol 2013;66: Philip T. Cagle et al. Pathology of the pleura: What the pulmonologists need to know Respirology (2011) 16, Tohru Tsujimura et al. Pathological and molecular biological approaches to early mesothelioma. Int J Clin Oncol (2012) 17: Aur_elien de Reynies et al. Molecular Classification of Malignant Pleural Mesothelioma: Identification of a Poor Prognosis Subgroup Linked to the Epithelial-to-Mesenchymal Transition. Clin Cancer Res; 20(5) March 1, P. M. Cury et al. The use of histological and mmunohistochemical markers to distinguish pleural malignant mesothelioma and in situ mesothelioma from reactive mesothelial hyperplasia and reactive pleural fibrosis. J. Pathol. 189: (1999) 9. Mark R. Wick et al. Immunohistochemical differential diagnosis of pleural effusions, with emphasis on malignant mesothelioma. Current Opinion in Pulmonary Medicine 2001, 7: Jenette Creaney et al. Comparison of mesothelin and fibulin-3 in pleural fluid and serum as markers in malignant Mesothelioma. Curr Opin Pulm Med 2015, 21: Shi Chen et al. The diagnostic value of survivin in malignant pleural effusion: A metaanalysis. Clinica Chimica Acta 441 (2015) Jenette Creaney et al. Pleural Fluid Mesothelin as an Adjunct to the Diagnosis of Pleural Malignant Mesothelioma. Disease Markers Volume A.M. Egan et al. Malignant pleural effusion. Q J Med 2014; 107:

9 14. P. Korczynski et al. Diagnostic utility of pleural fluid and serum markers in differentiation between malignant and non-malignant pleural effusions. Eur J Med Res (2009) 14 (Suppl. IV): C. Aleman et al., Differentiating between malignant and idiopathic pleural effusions: the value of diagnostic procedures. Q J Med 2007; 100: EVALUATION 1. Please mark the incorrect answer a. Malignant transudative effusions can occur in lung cancer with obstruction of the mainstem bronchus b. Blockade of lymphatic drainage may be invoked as one of the possible pathogenetic mechanisms of malignant pleural effusion c. Most of the pleural malignancies arise from tumor emboli to the visceral pleura, with secondary seeding to the parietal pleura d. Pleural fluid eosinophilia (> 10% eosinophils in the total nucleated e. cell count) is always diagnostic of malignant pleural effusion 2. Please mark the correct answer a. The yield from sending more than two separate pleural fluid specimens for cytology is very low b. In a MPE of unknown origin, a positive TTF-1 stain strongly suggests a primary non-small-cell lung cancer c. At least two mesothelial markers (e.g., calretinin, keratin 5/6, WT-1 protein) should be used to identified epithelial mesothelioma d. Mesothelin levels greater than 20 nm in effusions are highly suggestive of malignancy, particularly of malignant mesothelioma e. All the above 3. Please mark the correct answer a. Sensitivity of mesothelin for mesothelioma has been improved by using additional exclusion markers, such as carcinoembryonic antigen b. Survivin has been confirmed as a novel member of the inhibitor of apoptosis protein (IAP) family c. During tumorigenesis, Survivin expression is inversely correlated with apoptosis inhibition but positively correlated with proliferation and angiogenesis d. Survivin has been widely studied as a prognostic marker in NSCLC e. All the above 4. Please mark a true statement regarding malignant mesothelioma diagnosis a. Mesothelin pleural level has high sensitivity and specificity b. Low mesothelin rules out a malignant effusion c. Mesothelin level has low sensitivity and high specificity d. All the above 9

10 Malignant Pleural Effusion: which material for what disease Sebastian Fernandez-Bussy, MD 10

11 I have no real or perceived conflicts of interest that relate to this presentation: Affiliation/Financial Interest Grants/research support: Commercial Company Honoraria or consultation fees: Participation in a company sponsored bureau: Stock shareholder: Spouse/partner: Other support/potential conflict of interest: This event is accredited for CME credits by EBAP and EACCME and speakers are required to disclose their potential conflict of interest. The intent of this disclosure is not to prevent a speaker with a conflict of interest (any significant financial relationship a speaker has with manufacturers or providers of any commercial products or services relevant to the talk) from making a presentation, but rather to provide listeners with information on which they can make their own judgments. It remains for audience members to determine whether the speaker s interests, or relationships may influence the presentation. The ERS does not view the existence of these interests or commitments as necessarily implying bias or decreasing the value of the speaker s presentation. Drug or device advertisement is forbidden. 11

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13 Tumor markers seem to be a promising alternative and have been proposed to aid in the differentiation of the PE etiology. These include carcinoembryonic antigen (CEA), cancer antigens: CA- 125, CA 15-3, CA 19-9, CA 72-4, cytokeratin fragments (CYFRA 21-1), neuron specific enolase (NSA), and squamous cell carcinoma antigen (SCCA). However, the clinical value of these markers is still unclear. Eur J Med Res (2009) 14(Suppl. IV):

14 Pleural fluid (mean volume 50 ml) and blood samples for tumor marker measurements were collected during the first thoracentesis. They were centrifuged at 2000 rpm for 10 min and the supernatant was frozen at -20 C until assayed. Concentrations of CEA, CA- 125, CYFRA 21-1, and NSE were measured using electrochemiluminescence immunoassays on Roche Elecsys 1010 analyzer (Roche Diagnostics; Mannheim, Germany). Eur J Med Res (2009) 14(Suppl. IV):

15 The diagnostic utility was calculated for: 1) single tumor marker level in the pleural fluid (P) and serum (S); 2) single marker pleural fluid/serum level ratio (R); 3) a combination of four markers in the pleural fluid and serum. In the last case, the result was considered positive when the level of at least two markers was higher than the respective marker cut-off value. Eur J Med Res (2009) 14(Suppl. IV):

16 Eur J Med Res (2009) 14(Suppl. IV):

17 Eur J Med Res (2009) 14(Suppl. IV):

18 The median pleural fluid concentrations of the investigated tumor markers were significantly higher in malignant exudates compared with non-malignant effusions, with the exception of CA-125 which was similar. An analysis of the serum concentrations revealed comparable values of CA-125 and NSE in both groups, while the CEA and CYFRA 21-1 levels were significantly higher in malignant effusions. The pleural fluid/serum concentration ratios were similar for all markers in both investigated groups, with the exception of CEA which was significantly higher in the malignant group. Eur J Med Res (2009) 14(Suppl. IV):

19 Eur J Med Res (2009) 14(Suppl. IV):

20 Eur J Med Res (2009) 14(Suppl. IV):

21 CEA in PE proved to be the marker with the highest diagnostic accuracy. The area under curve (AUC) in the ROC analysis for this parameter was Previous studies which included this marker revealed the following AUC values: 0.97; 0.84, and 0.72 [3, 9, 10]. The diagnostic accuracy for serum measurements in our study was lower and equaled to Eur J Med Res (2009) 14(Suppl. IV):

22 CYFRA 21-1 is another marker with a high diagnostic yield, but contrary to the three other markers the serum, rather than plural fluid, level of CYFRA 21-1 shows a higher overall diagnostic accuracy. CYFRA 21-1, soluble fragments of cytokeratin 19, is expressed by all histological types of lung cancer, especially by squamous lung cancer [12]. Similarly to CEA, increased levels of CYFRA 21-1 in the pleural fluid may result from impaired lymphatic drainage or cancer involvement of the pleura. In our study, the AUC for this marker in serum was Eur J Med Res (2009) 14(Suppl. IV):

23 Studies in which lung cancer was the predominant cause of PE, revealed a higher diagnostic yield of this parameter. Serum CYFRA 21-1 has been found useful for the diagnosis of lung cancer, particularly of the squamous cell type. However, Dejsomritrutai et al [14] found that CYFRA 21-1 is a good marker also in a population of patients with PE caused by adenocarcinoma. These authors reported CYFRA 21-1 sensitivity of 81.5% and 74.1% in serum and the pleural fluid, respectively. The specificity of this marker was also high and reached as much as 97.1% for both serum and PE. Eur J Med Res (2009) 14(Suppl. IV):

24 We found a high sensitivity, but low diagnostic accuracy, for NSE. NSE is a known marker of small cell carcinoma. The sensitivity of pleural fluid NSE concentration was as high as 94.4%, while for the serum measurements it was 80.6%. However, the specificity of this marker was relatively low, thus the AUC reached 0.65 for pleural fluid and 0.62 for serum. Eur J Med Res (2009) 14(Suppl. IV):

25 CA-125 is particularly useful in detecting the recurrence of ovarian cancer [15], although the marker is also observed in malignancies of the endometrium, lung, breast, and gastrointestinal tract. In our patients, the accuracy of CA-125 measurement in PE and serum was 0.64 and 0.60, respectively. Eur J Med Res (2009) 14(Suppl. IV):

26 In the present study, a combination of four markers (CEA, CA-125, CYFRA 21-1, and NSE), with a cut-off for two or more markers, gave sensitivity and specificity of 91.7% and 86.8% for pleural measurements, 86.1% and 71.1% for serum, and 100% and 60.5% for the pleural fluid/ serum ratio, respectively. The AUC calculated from the ROC analysis achieved as much as 0.89, 0.82, and 0.88 for the pleural fluid, serum, and pleural fluid/serum ratio, respectively. It seems that the pleural fluid/serum marker ratio does not add any essential clinical information. A meta-analysis by Liang et al [16] showed similar results with the combinations of different tumor markers, e.g., the AUC for CEA/CA-125 was 0.86 and for CEA/ CYFRA Eur J Med Res (2009) 14(Suppl. IV):

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41 First, measurement of pleural CEA is likely to be a useful diagnostic tool for confirming MPE and is useful for the differential diagnosis between malignant pleural mesothelioma and metastatic lung cancer. A high level of pleural CEA seems to rule out malignant mesothelioma. Second, CA 15-3, CA 19-9, and CYFRA 21-1 are highly specific but insufficiently sensitive to diagnose MPE, and the combination of two or more tumor markers appears to increase the diagnostic sensitivity. Therefore, the results of tumor marker assays should be interpreted in parallel with clinical findings and with the results of conventional tests. Tuberc Respir Dis 2014;76:

42 Vascular endothelial growth factor (VEGF) as a diagnostic biomarker of MPE because of the high levels of VEGF present in MPE14. VEGF is thought to be the key mediator in the formation of MPE via increased vascular permeability and vascular leakage of fluid. A recent meta-analysis based on 1,025 patients in 10 studies concluded that VEGF might play a role in the diagnosis of MPE, while its diagnostic value is not satisfactory (Table 2)15. Mesothelin and fibulin-3 in pleural effusion have also been introduced as potential new biomarkers to detect pleural mesothelioma at an earlier stage. Tuberc Respir Dis 2014;76:

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44 Twenty-one studies with a total of 2,861 cases were included in present meta-analysis. The sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and DOR of CA 15-3 in the diagnosis of MPE were 0.58 [95% confidence interval (CI), ], 0.91 (95% CI, ), 8.93 (95% CI, ), 0.46 (95% CI, ) and (95% CI, ), respectively. In addition, the area under the curve (AUC) was In conclusion, due to the significantly high specificity of pleural CA 15-3 in detecting MPE, it may play a pivotal role in screening to identify patients who may benefit from further invasive pathologic examination, particularly in those presenting clinical manifestations of MPE but with negative cytological findings of the pleural fluid. However, ruling out MPE by testing CA15-3 alone is not recommended due to its limited sensitivity. 44

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47 The present meta-analysis suggested that the diagnostic value of CA 15-3 for MPE was far from perfection. Combining CA 15-3 with other markers may be an appropriate method for improving the diagnostic accuracy. The study by Romero et al found that the sensitivity and specificity of carcinoembryonic antigen combined with CA 15-3 in pleural fluid were 71% and 96%, respectively, which was better than testing CA 15-3 alone (15). Another study reported that the combination of thymidine kinase with CA 15-3 and procalcitonin appeared to be an optimal combination, nearly enabling differential diagnosing in all types of effusion 47

48 The diagnosis of malignant pleural effusion (MPE) is often challenging because differentiating MPE from benign effusion can be difficult using the currently available tools derived from thoracentesis; only approximately 50 70% of patients with MPE can be diagnosed by cytological examination of the pleural fluids [1]. Therefore, various tumor markers found in pleural effusions, including carcinoembryonic antigen (CEA), Cyfra 21-1, CA125, CA19-9, neuron-specific enolase, and squamous cell carcinoma antigen, have been investigated for their ability to differentiate malignant from benign pleural effusions [5 7]. However, the sensitivity of these tests is relatively low, and false-positive results have been described in almost all series. Thus, it is necessary to identify and evaluate more sensitive biological markers for MPE diagnosis [8]. The ideal marker would be office-based, rapid, and inexpensive and would have high sensitivity and specificity in the target population; this might reduce the burden and expense of frequent biopsies of the pleura. 48

49 Background: Survivin in pleural effusion is a promising marker for the diagnosis of malignant pleural effusion. Methods: Based on the principles and methods of Cochrane systematic reviews, PubMed, EMBASE, Web of Knowledge (ISI), the Cochrane Library, and China National Knowledge Infrastructure (CNKI) databases were searched to identify studies that assessed the diagnostic value of survivin in pleural effusion for malignant pleural effusion. Stata 12 and Meta-disc 1.4 software were used to test the heterogeneity and to perform the meta-analysis. Results: Our search returned 167 articles, ofwhich ten fulfilled the inclusion criteria. These studies included a total of 614 patients with malignant pleural effusion and 430 patients with benign pleural effusion as controls. The summary assessments revealed that the pooled sensitivity was 0.86 (95% CI: ) and the pooled specificity was 0.92 (95% CI: ). The positive likelihood ratio was 8.76 (95% CI: ), the negative likelihood ratio was 0.16 (95% CI: ) and the diagnostic odds ratio (DOR) was (95% CI: ). The area under the curve (AUC) for the pleural effusion survivin tests was , and the *Q index estimate for these tests was Conclusions: Survivin in pleural effusion has potential diagnostic value with advanced sensitivity and specificity and it can be used as adjunct tool for noninvasive diagnosis of malignant pleural effusion. 49

50 Survivin is a 12-amino acid protein (16.5 kda) that is located at chromosomal region 17q25. Survivin is an inhibitor of programmed cell death and mediates the suppression of apoptosis by inhibiting caspases 3 and 7, the terminal effectors in apoptotic protease cascades [10]. The survivin promoter is highly active in human tumor cells, but not in normal cells, and is up-regulated by hypoxia in tumors [11]. It is undetectable in normal differentiated tissues but is notably expressed in patients with lung cancer, breast cancer, bladder cancer, multiple myeloma, and lymphoma. 50

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59 Measurements. Pleural effusion samples were collected prospectively from 1331 consecutive patients. Mesothelin levels were determined by commercial ELISA in effusions and their relationship to concurrent pathology reporting and final clinical diagnosis was determined. Results pleural effusion samples from1331 individuals were analysed.the final clinical diagnosis was 183 MM, 436 non-mm malignancy, and 712 nonmalignant effusions. Effusion mesothelin had a sensitivity of 67% formm at 95% specificity. Mesothelin was elevated in over 47% of MM cases in effusions obtained before definitive diagnosis of MM was established. In the setting of inconclusive effusion cytology, effusion mesothelin had a positive predictive value of 79% for MM and 94% for malignancy. Conclusions. A mesothelin-positive pleural effusion, irrespective of the identification of malignant cells, indicates the likely presence of malignancy and adds weight to the clinical rationale for further investigation to establish a malignant diagnosis. 59

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62 Purpose of review Malignant mesothelioma is an asbestos-induced, aggressive tumour, which frequently presents with pleural effusion. There are over 60 reported causes that can result in the development of a pleural effusion. Currently, there are no tumour biomarkers in widespread clinical use for the differential diagnosis of mesothelioma from other diseases. With the incidence of mesothelioma expected to continue to increase, it is timely to review the current status of effusion-based biomarkers for mesothelioma diagnosis. Recent findings The majority of recent studies have evaluated soluble mesothelin in effusions in a diagnostic setting for mesothelioma. However, at high specificity, the sensitivity of the assay is limited to approximately 60% at the time of diagnosis. There is considerable research effort directed toward the identification of new markers for mesothelioma through a variety of genomic, proteomic and immunomic based platforms. One of the few new biomarkers to be identified through a biomarker discovery pipeline and evaluated in pleural effusions is fibulin-3. Preliminary results on the diagnostic accuracy of fibulin-3 have been inconsistent. Summary To date, soluble mesothelin remains the best available biomarker for mesothelioma and a positive result is clinically useful in patients with pleural effusions in whom the diagnosis is uncertain. 62

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64 At a histopathologic level, there are essentially four circumstances in which immunophenotypic study plays an important role in the recognition of mesothelioma. (1) Mesothelioma versus adenocarcinoma (2) Sarcomatoid mesothelioma versus primary or metastatic pleural sarcoma versus metastatic sarcomatoid Carcinoma (3) Mesothelioma versus reactive mesothelial hyperplasia (4) Desmoplastic sarcomatoid mesothelioma versus fibrous pleuritis 64

65 Each of the above-cited diagnostic questions requires a different panel of immunohistochemical reagents. In cases of suspected spindle-cell or desmoplastic mesothelioma, these should include antibodies to keratin and calretinin; other markers are necessary only to subtype truly mesenchymal keratin-negative neoplasms. Recently, antibodies to cytokeratins 5/6 have been advocated as contextually specific markers for mesothelioma. Ordoñez found that 40 cases of mesothelioma were positive for keratins 5/6, whereas adenocarcinomas of the lung were negative. Nevertheless, the selected analyses have also found that 11 to 14% of nonpulmonary adenocarcinomas show focal reactivity for cytokeratins 5/6. Cytokeratins 7, 8, 18, and 19 are seen in all mesotheliomas and adenocarcinomas, whereas cytokeratins 5, 6, 14, and 17 are observed in some mesotheliomas but not adenocarcinomas. Unfortunately, the latter markers are absent in sarcomatoid tumors. 65

66 Carcinoembryonic antigen: Virtually all pleural mesotheliomas lack CEA, whereas the vast majority of pulmonary adenocarcinomas express that marker. CD15 has a high level of specificity for adenocarcinoma among all epithelial tumors. Nevertheless, occasional mesotheliomas (usually primary peritoneal lesions) may show focal staining for this marker. B72.3 This antibody recognizes a widely distributed epithelial determinant (tumor-associated glycoprotein-72), which is a cell-membrane glycoprotein. Irrespective of their sites of origin, a majority of adenocarcinomas show reactivity with B72.3. Ber-EP4 is another generic epithelial marker that was originally thought to be specific for adenocarcinomas. It is now apparent that roughly 20% of mesotheliomas demonstrate Ber-EP4 reactivity as well. 66

67 Calretinin is a cytoplasmic and nuclear calcium-binding protein. In contrast to most antibodies used for the evaluation of possible mesotheliomas, labeling for calretinin represents positive support for that diagnosis. This marker is present in more than 85% of mesotheliomas of the epithelial type. The authors own experience is that the majority of sarcomatoid lesions are, in fact, positive for calretinin. 67

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72 Aquaporin-1 (AQP1) is a molecule involved in the growth of MM cells, and yet is a factor reported to correlate with improved survival rates for MM with an epithelioid component, in comparison to AQP1-poor MM, as assessed from AQP1 expression by epithelioid MM cells only (apart from co-expression by stromal endothelial cells in addition to the tumour cells). Prognostic factors for MM include not only the histological subtypes, but other independent variables that include (among others), AQP1 expression by mesothelioma cells, the clinical status of the patient, the serum neutrophil:lymphocyte ratio and blood thrombocytosis 72

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77 Mesothelial Hyperplasia Vs Epitheliod Mesothelioma Husain A., et al., Arch Pathol Lab Med 2013;137:

78 Epitheliod Mesothelioma Vs Lung AdenoCa Husain A., et al., Arch Pathol Lab Med 2013;137:

79 Sarcomatoid Mesothelioma Vs SCC and TCC Scherpereel A, et al. Eur Respir J 2010;35:

80 Fibrous Pleurisy vs Sarcomatoid Mesothelioma Husain A., et al., Arch Pathol Lab Med 2013;137:

81 Benign Vs Malignant 81

82 How to send your material to pathology Prof. Dr Philipp A. Schnabel Instit. Alllgemeine&Spezielle Pathologie University of Saarlandes Homburg/Saar GERMANY 82

83 Interpretation of the results in the clinic Prof. Arnaud Scherpereel Pulmonary and Thoracic Oncology Department Hôpital Calmette University Hospital of Lille (France) INSERM Unit 1019 Pasteur Institute of Lille 2 Avenue Oscar Lambret Lille FRANCE arnaud.scherpereel@chru-lille.fr 83

84 MALIGNANT PLEURAL EFFUSION MANAGEMENT ERS 2015 Congress Amsterdam (Netherlands) PG 10 Session : Malignant pleural effusion management : From standard cytopathology to molecular biology Saturday, 26 September 2015 Pr Arnaud SCHERPEREEL Pulmonary and Thoracic Oncology Department Hôpital Calmette University Hospital of Lille (France) INSERM Unit 1019 Pasteur Institute of Lille arnaud.scherpereel@chru-lille.fr 84

85 Financial disclosures I have no conflict of interest for this lecture 85

86 Let s talk about Pathogenesis, Diagnosis and Management of: Malignant pleural effusions (MPE), pleural metastases and malignant pleural mesothelioma (MPM) *Ref: chapter Pleural and chest wall tumours A Scherpereel. ERS Handbook

87 Pleural Malignancies Clinical presentation = usually pleural effusion Malignant pleural effusion (MPE) found in 6% of cancers history; reveals the cancer in half cases But in necropsy series: pleural effusion is only found in half of pleural tumors Dyspnea: most frequent symptom; other NON specific signs: cough, chest pain, asthenia and weight loss, more rarely fever, pneumothorax Light RW & Lee YCG. Textbook of Pleural Disease, 2 nd ed

88 Malignant pleural effusion (MPE) patients / year in the USA; incidence: - Breast Cancer: 1 /3 - Lung Cancer: 1 /4 (most common etiology >40% of cases) - MPM: 95% (unilateral+++) Lung, breast, ovary and gut cancers and lymphoma = 80% of MPE Primitive malignancy: not found in 10% of cases 88

89 Pathogenesis of malignant pleura Still partly unknown 89

90 Pathogenesis of malignant pleura Pleural tumor involvement may result from different mechanisms: 1. A blood dissemination or more often from tumor emboli to the visceral pleura with secondary seeding to the parietal pleura (main mechanism for lung cancer) 2. direct invasion from adjacent structures (lung, chest wall...) by the tumor (lung or breast cancer) 3. Blood metastases to the parietal pleura from tumors other than lung cancer 4. lymph vessels spreading 5. Effusion may be due to the pleural tumor lesions or to a lymphatic blockade at the mediastinal level Sahn SA. Malignant pleural effusions. Pleural diseases. Philadelphia : WB Saunders, 2004 :

91 Pathogenesis of malignant pleura (2) MPE depend also on interactions between tumor cells and mesothelial cells through growth factors such as VEGF that increase vascular permeability and angiogenesis. Finally, MPE need several pathogenic factors such as: Increase of capillaries permeability Lesions of the lymphatic system draining the pleural space (pores of Wang at the postero-basal parietal pleura) or at the level of the thoracic channel Pericardial involvement 91 Sahn SA. Malignant pleural effusions. Pleural diseases. Philadelphia : WB Saunders, 2004 :

92 Pathogenesis of malignant pleura (3) CAUTION : a pleural effusion is NOT systematically synonym of MPE in the context of tumor because it may be induced by other mechanisms : pneumonia and/or atelectasia due to bronchial obstruction transudate induced by severe denutrition or cardiac failure or even a drug (MTX, CPM ) or radiotherapy-induced effusion (breast cancer+++) Therefore the strategy (diagnosis/ttt) may differ whether the patient has a cancer background or not, but should always rely on cytology or better on histology+++ 92

93 Prognosis of MPE and pleural metastases Poor prognosis (one year MS = 13%) However survival may greatly vary according to the primary cancer: lung cancer << breast cancer or lymphoma New 2009 TNM staging of NSCLC: MPE (or pericardial effusion) or pleural metastases = stage T4 (TNM 1997) NOW STAGE M1a (IV) Survival at 5 years of tumors ct4n0m0 by pleural involvement =2% (similar to M1 patients) vs by other cause = 25% (p = 0,0001) Journal of Thoracic Oncology 2007; 2 :

94 Malignant (Pleural) Mesothelioma Very agressive tumor issued from mesothelial cells mostly of the pleura (MPM : 80% of the cases) Bad prognosis : median survival = 9-12 months 94

95 Diagnostic issues in malignant pleural diseases: a specific clinical profile? Pleural effusion +++ Pleural thickening Pneumothorax (MPM, underlying pulmonary disease ) Non-specific signs: dyspnea, cough, chest pain, asthenia and weight loss 95

96 Management of MPE and MPM MPE: please refer to ERS Handbook MPM: refer to ERS/ESTS Guidelines Scherpereel et al, ERJ 2010 (ongoing update by European Taskforce) 96

97 Clinical and/or radiological diagnosis of unilateral Pleural Effusion: MPE? Previous asbestos exposure? Clinical and/or radiological suspicion of MPM Small effusion, hemostasis disturbancies = relative contra-indications for ultra-sound (US)-guided procedure by trained physicians Thoracocentesis for diagnosis and drainage Proceed to MPM algorythm Exsudate Light s Criteria Transudate: seak and treat cause of transudate Cytology: tumor cells? NO If no argument for tuberculosis, infection YES NO Previous diagnosis of malignancy (lung, breast )? YES Chest CT scan mandatory if no contralateral mediastinal shift on chest Xray bronchoscopy if main bronchus stenosis is suspected: trapped lung? Is pleural histology needed for diagnostic clarity? Otherwise start treatment 97

98 Chest CT scan mandatory if no contralateral mediastinal shift on chest Xray bronchoscopy if main bronchus stenosis is suspected: trapped lung?! Blind pleural biopies if suspicion of tuberculosis? Thoracoscopy Surgery at risk Contra-indications Thoracoscopy Pleural symphysis In the same time if large effusion AND bulky tumor lesions of the pleura Histological proof of pleural metastases CT scan or US -guided transthoracic biopsies Mini-thoracotomy for pleural biopsies Pleurodesis (Talc poudrage ) also if reccurent pleural effusion; alternatives: talc slurry, undwelled catheter Treatment of the primary cancer (chemotherapy and/or hormone therapy and best supportive care (BSC)...) 98

99 Medical Thoracoscopy or VATS: Gold standard diagnostic procedure Sensitivity : 95% Specificity : 100% Complications (non lethal) : < 10% 99

100 MPE aspect in thoracoscopy (Source : Pr A. Scherpereel and Dr Ph. Ramon) 100

101 Thank you for your attention Welcome to join the European Respiratory Society group on Pleural and Mediastinal malignancies (11.2) of the Thoracic Oncology Assembly (N 11) +++! 101

102 SUMMARY Atypical mesothelial hyperplasia and other pitfalls in MPE diagnosis Prof. Françoise Galateau-Salle Centre Leon Bérard Lyon University Hospital Caen Avenue de la Côte de Nacre Cedex 05, Caen FRANCE Atypical mesothelial hyperplasia happens in response to a great variety of benign or malignant processes including infectious pleuritis, pneumothorax, underlying pneumonia, connective tissue diseases (rheumatoid arthritis and systemic lupus erythematosus), pulmonary embolism, drug reactions, and asbestos associated effusions or in association with underlying bronchopulmonary carcinoma. Moreover, exudative pleural effusion in most cases is the initial symptom revealing a mesothelioma or a metastasis as the presenting site of another cancer. According to the literature 50% of patients with a metastatic disease develop pleural effusion and 75% of them are symptomatic. In the majority of the case the primary site is found with in order of frequency from Antunes et al (1, 2): lung, breast, ovarian, lymphoma, GI, GU tract, mesothelioma. In 10% of the cases the primary site remains unknown (2). It also may represent a precancerous step or the early phase of a malignant mesothelioma. Consequently, the separation between benign mesothelial superficial proliferation and a malignant mesothelioma in a patient with a unilateral pleural effusion is crucial for the patient care and is extremely challenging for both clinician and pathologist. On the pathologist side, a first look on cell morphology, raise several questions; is the sample significant enough for an accurate diagnosis???, Is the process malignant??? Then when the process looks malignant, the followed questions; is the lesion a malignant mesothelioma or another tumor mimicking a mesothelioma. Carcinoma may superficially metastases to the surface of the pleura closely mimicking a malignant mesothelioma. It is well known that sarcomatoid mesothelioma and other spindle cell tumors located to the pleura do not peel in the pleural cavity. This course will focus on epithelioid mesothelioma. Indeed, the diagnostic approach in these situations is dependent on the clinical presentation and on the material sent to the pathology lab. 1. The separation between benign mesothelial proliferations on routine biopsies has been largely described by Churg et al(3-6), and new updates were described in the 2015 WHO classification. Two major scenarios can arise for the pathologist: a. The lesion appears on cytology or tissue biopsy specimens of epithelial/epithelioid type the question is this? i. A reactive mesothelial hyperplasia mimicking a malignant process. ii. If malignant; is the lesion a malignant mesothelioma, a metastasis of a carcinoma/ or of a melanoma. iii. Or a so-called well differentiated superficial papillary mesothelioma (rare mésothéliale tumor of uncertain behavior). b. The lesion is morphologically a spindle cells/fibroblastic proliferation (spindle cells do not peel in the serosal cavity) on tissue biopsy is this? i. A reactive cellular diffuse proliferation (chronic fibrous pleuritis) ii. A rare fibromatois desmoid invading the pleura iii. If malignant; is the lesion a sarcomatoid/desmoplastic mesothelioma versus a metastasis of a sarcomatoid carcinoma or a sarcoma? 102

103 2. The lesion looks malignant the issue to reach a diagnosis;iscytology orcore tissue biopsy or open surgical biopsy the best approach?? 3. Facing these situations, 1. the clinical presentation can help the pathologist in separating a metastasis versus a mesothelioma 2. What are the important elements brought by every practitioner for a quality and safe diagnosis? 3. What is the role in 2015 of ancillary techniques to solve the problem of the separation benign from malignant? 4. How can we avoid pitfalls?? Nowadays, even if pathologists better know how to recognize a malignant mesothelioma on morphology or a metastasis in many casesto separate benign versus malignant mesothelial proliferation in a significant proportion of cases may be extremely challenging. The role of this course is to consider the management of pleural effusion from standard cytopathology to molecular biology and to evaluate if the transition, in 2015, is real in routine practice. The diagnostic approach Pathologist side: When confronted with a pleural process for cytological or histological diagnosis it is essential for the pathologist to know as much as possible of the clinical and radiologic information when they are available. For example it is important for the pathologist to know if it is a free floating pleural effusion without thickening and nodularity (could be a WDPM) or if it is a diffuse process (diffuse pleural thickening with or without nodularity) could be a connective tissue disease or a malignant mesothelioma desmoplastic type, or a mass located in the lung or in the thoracic wall (Sarcomatoid carcinoma versus localized sarcomatoid mesothelioma). It is also important to know the age and the sex of the patient, the context of previous history of cancer, due to secondary tumors affecting the serosal cavities that can be great mimickers of mesothelioma and force to check the expression of organ specific markers for the differential diagnosis (ie ER receptors in women to detect breast metastasis). Clinician side: The technique of the pleural effusion aspiration is crucial and determines the quality of the pre-analytical phase, but is mainly dependent on the clinical presentation, the comorbidities, the experience of the clinician. In the majority of the cases pleural aspiration is the initial step to relieve the patient from dyspnea. The sample should be sent as soon as possible to the pathology lab to avoid autolysis. Unfortunately due to undiagnosed exudative pleural effusion pleuralbiopsy is necessary. In 2015, thoracoscopy and CT-guided core biopsy have a quite similar diagnostic yield for the diagnosis of a malignant diseases including mesothelioma with a reported sensitivity of 76-88% and up to 100% specificity depending of the authors series (1, 2). In order to help, the quality of cytopathological examination, if a biopsy is taken or cell blocks made, the sample should be fixed immediately and preserve in formalin 5%. It is also important to avoid acid fixative (AFA and others new fixatives) as it is responsible of absence of signal by FISH analysis. Even though, an open surgical biopsy is a better material for the accurate diagnosis of subtyping mesothelioma 83% versus 74% on VATS and 44 % with CT-guided biopsy (8, 9). On the pathologist side Cytology samples are the initial and major step to approach the diagnosis of a pleural effusion. The cytologist, without major difficulties, recognizes malignant effusions and many studies have shown that malignant effusions are diagnosed in nearly 60 % by cytological examination alone, but drop down to 30% 103