Prof. Dr. Metin ÖZKAN

1. New Biochemical and Immulogical parameters in Differential Diagnosis of Malign and benign Pleural Effusions Prof. Dr. Metin ÖZKAN

2. I have no conflict of interest related to content of this presentation

3. Diagnosis of malignant pleural effusions (MPEs) • Based on the finding of tumor cells in pleural fluid • pleural fluid cytology is positive in only 60% of cases • Approximately 50% of patients with cancer will develop pleural effusion during the course of the disease. • 15% of all pleural effusions are related to malignancy • More than 75% of MPEs are caused by carcinomas of the lung, breast or ovary, or by lymphomas, with metastatic adenocarcinoma • Mesotheliomas are primary cancers of pleural lining cells.

4. Diagnosis of malignant pleural effusions (MPEs) • The gross appearance: haemorrhagic (MPE) or opalescent (paraneoplastic chylothorax) • Half of bloody effusions are malignant, only 11% of MPEs are bloody • Unexplained exudate suggests the possibility of a MPE, although exudates may be caused by non-neoplastic inflammatory conditions. • 3% to 10% of MPE are transudative in nature, the absence of exudative characteristics does not exclude the possibility of either a MPE or a paraneoplastic effusion.

5. Diagnosis of malignant pleural effusions (MPEs) • The majority of MPEs are lymphocyte predominant • Eosinophil >10% : benign? • 2% - 24% of eosinophilic effusions may be malignant • Amylase may be elevated in MPE: Routine measurement? • The proposed cut-off point 100 IU/L and 200 IU/L. • Subsequent studies indicate that only 1–8% of pleural effusions are amylase rich, • High pleural fluid amylase associated with shorter survival

6. Diagnosis of malignant pleural effusions (MPEs) • Pleural fluid pH < 7.30 and glucose < 60 mg/dL in 1/3 of MPE. • Decreasing pH and glucose : Shorter suvival and failed pleurodesis. • Pleural fluid pH and glucose can be used for selecting patients for pleurodesis ?? • Subsequent metaanalyses, however, demonstrate that pH and glucose have limited value for predicting either survival or pleurodesis outcome.

7. Diagnosis of malignant pleural effusions (MPEs) • Standard cytological evaluation: diagnostic yield; 62% - 90% • Wide variation in effusions due to non-Hodgkin’s lymphoma (22–94%). • Increased number of thoracenteses –increased diagnosis • Standard pleural fluid cytology has difficulties with specificity because of limitations in differentiating between adenocarcinoma, mesothelioma, lymphoma and non-malignant reactive lymphocytosis.

8. Diagnosis of malignant pleural effusions (MPEs) • Accurate diagnosis of the cause of a pleural effusion can be challenging. • Analysis of soluble biomarkers from effusions may be a useful adjunctive. • Ideal biomarkers should be both sensitive and specific to the disease state being examined • The value of pleural fluid tumor markers in diagnosing malignancy remains limited because further pathologic examination is warranted

9. Diagnosis of malignant pleural effusions (MPEs): tumour markers • Biomarker: a biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease, such as cancer, infection, or HF. • Ideal biomarker: • easily measured at a reasonable cost (analytical validity), • must provide information that is not already available from a routine clinical assessment (clinical validity), • eventually, should aid in decision making (clinical usefulness)

10. Tumour markers • CEA is the most common marker to have beenstudied ; increased in  colorectal, gastric, pancreatic, lung and breast carcinoma. • CEA levels are higher in smokers, inflammatory bowel disease, peptic ulcer disease, pancreatitis, biliary disease, liver dysfunction and hypothyroidism • Carbohydrate antigen (CA)125 is a tumour-associated antigen commonly seenin ovarian carcinoma and is used to assess theresponse to chemotherapy and for early detectionof relapse. • CA 19-9 is a tumourantigen whose level increases particularly ingastrointestinal tumours • CA 15-3 is increased in breast cancer. • CYFRA 21-1 is afragment of cytokeratin 19 which provides a usefulmarker for epithelial malignancies, distinctlyreflecting ongoing cell activity.

11. Carcinoembryonic antigen (CEA)

12. Meta-analysis of 45 studies • Sensitivity 0.54, specificity 0.94, diagnostic odds ratio 22.5 • Analysis of a subset of 11 studies in ruling out malignant mesothelioma : sensitivity and specificity of a CEA level ; 0.97 (95% CI: 0.93–0.99) and 0.60 (95% CI: 0.55–0.65), respectively. Conclusions: Measurement of pleural CEA is likely to be a useful diagnostic tool for confirming MPE, and is also helpful in the differential diagnosis between malignant pleural mesothelioma and metastatic lung cancer

13. 416 patients • 166 malignant, • 77 probable malignant • 173 benign effusions) • Cutoff points that yielded 100% specificity (ie, all patients with benign effusions had levels below this cutoff)

14. Results: • MPEs had higher values • At 100% specificity, pleural • CEA > 50 ng/mL, (sensitivity: 29%) • CA 125 > 2,800 U/mL, (sensitivity: 17%) • CA 15–3 > 75 U/mL, (sensitivity: 30%) • CYFRA 21–1 > 175 ng/mL (sensitivity: 22%) • Combined 4 TM markers increased sensitivity to 54%, • Combined TM markers + cytology: increased 18% • More than 1/3 of cytology-negative malignant PEs could be identified by at least one marker of the panel. Conclusions: a tumor marker panel may represent a helpful adjunct to cytology in order to rule in malignancy as a probable diagnosis,

15. The role of tumour markers such as carbohydrate antigen (CA) 125, CA 15-3, CA 19-9 and CYFRA 21-1 (a fragment of cytokeratin 19) in differentiating malignant pleural effusions (MPE) from benign effusions

16. 29 studies met the inclusion criteria for the analysis. • Sensitivity and specificity of these tumour markers: • CA 125, 0.48/0.85; • CA 15-3, 0.51/0.96; • CA 19-9, 0.25/0.96; • CYFRA 21-1, 0.55/0.91 • When two or more tumour markers were combined, or combined with CEA, the sensitivity and specificity were all increased Not recommended using one tumour marker alone, but the combination of two or more tumour markers seems to be more sensitive

17. 22 studies were summarized based on their discussions on the result of the CYFRA 21-1 assay in pleural effusion and differential diagnosis evaluation in the Chinese population The eligible studies included 1653 MPE cases and 1011 BPE controls.

18. The CYFRA 21-1level was significantly higher in MPE (n1653) than that inBPE (n1011) (p<0.00001). (all 22 studies) • The CYFRA 21-1 level in pleural effusion (13 studies) was significantly higher than that in serum (p < 0.00001). • The CYFRA 21-1 level in the pleural effusion was higher in SCC (n268) patients than thatin adenocarcinoma patients (n320) p < 0.00001. (8 studies) • The sensitivity of CYFRA 21-1 reported in the articles ranged from 46% to 94%, and the specificity ranged from 57% to 100%. • The measurement of pleural CYFRA 21-1 is likely to be a useful diagnostic tool for the confirmation of MPE.

19. J Clin Lab Anal. 2007;21(6):398-405. Diagnostic utility of pleural fluid carcinoembryonic antigen and CYFRA 21-1 in patients with pleural effusion: a systematic review and meta-analysis. Gu P, Huang G, Chen Y, Zhu C, Yuan J, Sheng S. • Studies for differential diagnosis of PEs by pleural fluid CEA and CYFRA 21-1 were collected. • A total of 19 studies were included in the meta-analysis, with a total of 3,228 subjects. • Pooled sensitivity and specificity of CEA and CYFRA 21-1 were 45.9% (43.2-48.5%) and 97.0% (96.0-97.8%), and 47.3% (44.0-50.6%) and 91.8% (89.5-93.7%), respectively. • Both CEA and CYFRA 21-1 have a threshold effect, the main source of heterogeneity was from variable assay methods. • Both CEA and CYFRA 21-1 have good performance in the differential diagnosis of PE, when compared with CEA, CYFRA 21-1 has no advantage.

20. Biomarkers for Mesothelioma • Malignant mesothelioma has a very dismal prognosis with very few patients surviving one year after diagnosis. • A reliable biomarker would be very helpful • Old markers such as hyaluronic acid, CYFRA 21.1, and other cancer antigens (CA 15.3, CA 125 or CA 19.9 or CEA) are not sensitive or specific enough and cannot be used in practice. • More recently new molecules, such as soluble mesothelin, osteopontin, and fibulin 3 have been proposed for diagnostic purposes

21. Mesothelin • 40 kDa membrane-bound protein attached to the mesothelial cell surface by phosphatidylinositol • Most extensively investigated marker. • It can be measured by ELISA in either serum or pleural fluid.

22. Soluble mesothelin related peptides (SMRPs) • Mesothelin: • Soluble Mesothelin: A soluble protein in serum captured using antibodies targeting mesothelin • SMRPs Released into the circulation from malignant cells that overexpress mesothelin Hassan R, Bera T, Pastan I.Clin Cancer Res 2004; 10:3937–3942. Maeda M, Hino O. Pathol Int 2006; 56:649–654.

23. Prospective study, SMRPs in serum and pleuraleffusion; mesothelioma (n74), pleural metastasisof carcinomas (n 35), or benign pleural lesions associated withasbestos exposure (n 28), • Findings: Mean serum SMRP level was higher in patients with mesothelioma(2.05 ± 2.57 nM/L thanin patients with metastasis (1.02 ± 1.79 nM/L) or benign lesions(0.55 ± 0.59 nM/L). • Serum SMRP was 0.872 for differentiatingmesothelioma and benign lesions, cut-off0.93 nM/L (sensitivity80%, specificity 82.6%). • Serum SMRP differentiatingmetastasis and mesotheliomawas 0.693, cut-off1.85 nM/L (sensitivity58.3%, specificity 73.3%). • SMRP values in pleural fluidwere higher than in serum in all groups(mesothelioma: 46.1 ± 83.2 nM/L; benign lesions: 6.4 ± 11.1 nM/L; metastasis: 6.36 ± 21.73 nM/L).

24. (A) Serum SMRPs in differentiating MPM and BPLAE. (B) Serum SMRPs in differentiating patients with MPM and carcinoma. (C). Pleural SMRPs in differentiating MPM and BPLAE. (D) Pleural SMRPs in differentiating patients with MPM and carcinoma.

26. Ann Thorac Surg 2008;85:265–72 • The MPM pleural effusionSMRP was significantly higher than benign or othernon-MPM pleural effusions • (65.57 11.33 nM vs 27.4611.25 nM [p 0.003] and 18.99 7.48 nM [p 0.044],respectively).

28. Is SMRP an ideal biomarker? The sensitivity and specificity of SMRPs: 77%-76% in a multicenter study from France 67%-98% in a Western Australian cohort of 234 patients The diagnostic accuracy of pleural fluid SMRP was as good as serum SMRP Scherpereel A, et al. Am J Respir Crit Care Med 2006; 173:1155 Creaney J, et al.Thorax. 2007 ;62:569-76.

29. Is SMRP an ideal biomarker? False positive: high in ovarian, pancreatic lung CAs and non-Hodgkin’s lymphoma False negative:negative in all sarcomatoid and in up to one half of epithelioid mesothelioma Scherpereel A, et al. Am J Respir Crit Care Med 2006; 173:1155 Creaney J, et al.Thorax. 2007 ;62:569-76. B.D. Grigoriu BD, et al. Clinical utility of diagnostic markers for malignant pleural mesothelioma. Monaldi Arch Chest Dis 2009; 71: 1, 31-38.

30. Can we use SMRPs in populations exposed to asbestosis? • 535 subjects who had been exposed to asbestos or silica • The strongest independent (non-cancer) factors on SMRP levels were age and glomerular filtration rate (GFR) • SMRP levels were also affected by smoking • SMRP demonstrated poor sensitivity (15.1%) for SMRP values among subjects with non-malignant asbestos-related disorders was found when compared to currently healthy subjects with a history of asbestos exposure. • Park EK, et al. A predictive equation to adjust for clinical variables in soluble mesothelin-related protein (SMRP) levels. Clin Chem Lab Med 2012 Dec;50(12):2199-204.

31. Summary • SMRP seems to have the best sensitivity and specifity to diagnose mesothelioma • High in ovarian, pancreatic and lung CA • Low in sarcomatoid type • Not recomended for screening • Need for an equation based on age and GFR to improve its diagnostic accuracy, • SMRP can be used to support diagnosis

32. Fibulin-3 Fibulin-3 is an extracellular glycoprotein that mediates cell-to-cell interactions and has variable angiogenic effects

34. Plasma Fibulin-3 levels: • 92 mesothelioma, • 136 asbestos-exposed persons without cancer, • 93 effusions not due to mesothelioma, • 43 healthy controls, • Pleural effusion Fibulin-3 levels • 74 mesothelioma, • 39 benign effusions, • 54 with malignant effusions not due to mesothelioma) • Tumor tissue was examined for fibulin-3 by immunohistochemical analysis,

35. Plasma fibulin-3 levels were significantly higher in patients with pleural mesothelioma (105±7 ng/ml: Detroit cohort and 113±8 ng/ml: New York cohort) than in asbestos-exposed persons without mesothelioma (14±1 ng per milliliter and 24±1 ng per milliliter, respectively; P<0.001). • Effusion fibulin-3 levels were significantly higher in patients with pleural mesothelioma (694±37 ng per milliliter in the Detroit cohort and 636±92 ng per milliliter in the New York cohort) than in patients with effusions not due to mesothelioma (212±25 and 151±23 ng per milliliter, respectively; P<0.001). • Fibulin-3 stained tumor cells in 26 of 26 samples

41. Biomarkers for Mesothelioma:Osteopontin Serum osteopontin, have little value in distinguishing mesothelioma from metastatic pleural carcinomas or benign asbestos pleural diseases but may be used as a prognostic marker. Scherpereel A, Lee YCG. Curr Opin Pulm Med 2007;13:339–343 Grigoriu BD et al. Cancer Res 2007 May 15;13(10):2928-35.

42. Retrospective study; CEA and CYFRA 21-1 were studied in pleural effusion of unknown origin in patients who had undergone medical thoracoscopy Results: 134 patients (103 men and 31 women); 33 had MPM. The level of pleural effusion CYFRA 21-1 and the CYFRA 21-1/CEA ratio were significantly different between MPM and other diseases (p<0.01). The sensitivity and specificity of the pleural effusion CYFRA 21-1/CEA ratio were 84.8% and 80.2%, respectively, when the CYFRA 21-1/CEA ratio cut-off value determined by receiver operating characteristic curve analysis was 19.1. Conclusion: MPM should be suspected when the CYFRA 21- 1/CEA ratio in pleural effusion is greater than 19.1.

43. Figure 1. Receiver operating characteristic curve of the ratio cytokeratin 19 fragment (CYFRA 21-1)/carcinoembryonic antigen (CEA) in pleural effusion distinguishes mesothelioma from other malignant diseases. CYF, CYFRA 21-1; Sens, sensitivity; Spec, specificity. Figure 2. Receiver operating characteristic curve of cytokeratin 19 fragment (CYFRA 21-1) in pleural effusion distinguishes mesothelioma from other malignant diseases in patients with carcinoembryonic antigen (CEA) <5 ng/ml.

44. Immunocytochemical Markers • Immunocytochemical staining on cell block preparations assists in establishing MM and metastatic adenocarcinoma discrimination through the application of panels consisting of a variety of antibodies • Ideal antibody combination?? • a sensitivity of about 80% is desirable for inclusion in the panel. • To confidently validate the diagnosis of epithelioid mesothelioma as opposed to adenocarcinoma, 2 positive mesothelioma markers along with 2 negative adenocarcinoma markers are required • The panel can be expanded if the results are inconclusive. • initially use small panels, which include epithelial membrane antigen (EMA), calretinin, CEA, and thyroid transcription factor-1 (TTF-1). Clin Chest Med 34 (2013) 27–37 http://dx.doi.org/10.1016/j.ccm.2012.11.002

45. to establish the primary site of a malignant effusion in patients with an occult primary or multiple primaries. • For example, determination of the primary origin of adenocarcinomas, the most commonly found malignancy in effusion samples, may necessitate the use of the following immunocytochemical markers: • TTF-1 (lung cancer), • cytokeratin 7 (upper gastrointestinal and pancreatobiliary tract), • cytokeratin 20 (colorectal) and, • in female patients, • estrogen receptors (breast, female genital tract), • mammaglobin (breast), • Wilms tumor gene 1 (WT-1; female genital tract). Clin Chest Med 34 (2013) 27–37 http://dx.doi.org/10.1016/j.ccm.2012.11.002

46. Clin Chest Med 34 (2013) 27–37 http://dx.doi.org/10.1016/j.ccm.2012.11.002

47. Clin Chest Med 34 (2013) 27–37 http://dx.doi.org/10.1016/j.ccm.2012.11.002

48. TTF-1 is a sensitive and very highly selective marker ofadenocarcinoma of pulmonary origin in cytologic preparationsand also is effective in the distinction between malignant mesothelioma and pulmonary adenocarcinoma in pleural fluids..

49. Fluorescence in situ hybridization • Aneuploidy is commonly found in cells of solid tumours. • Fluorescence in situ hybridization analysis, image analysis cytometry and PCR are relatively simple and robust techniques for detecting aneuploidy and are applicable to small tissue samples. • Detection of aneuploidy contributes to pleural fluid analysis for detecting MPE and leads to meaningful therapeutic opportunities? • FISH (fluorescence in situ hybridization) is a cytogenetic technique that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. • FISH uses fluorescent probes that bind to only those parts of the chromosome. • Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. • FISH is often used for finding specific features in DNAbut also can be used to detect and localize specific RNA targets (mRNA, lncRNA and miRNA) in cells, circulating tumor cells, and tissue samples. Rosolen DCB, et al. Efficacy of two fluorescence in situ hybridization (FISH) probes for diagnosing malignant pleural effusions. Lung Cancer (2013), http://dx.doi.org/10.1016/j.lungcan.2013.02.001

50. Thisstudydemonstratedthatcytogeneticanalysis of freshpleuralfluidsamplesusingonlytwo FISH probestodiagnoseMPEsparticularly in caseswhichcytology is inconclusive. • For FISH, a pleuralfluidsamplewascentrifuged at 1400 rpmfor 5 min, andthesedimentwasfixed in freshCarnoy’ssolution (methanol: aceticacid, 3:1) forslidepreparation. • Theslideswerethenhybridizedwithcentromereprobesforchromosome 11 (redstaining) andchromosome 17 (greenstaining) • Thedigitalimageswerecapturedwith an Olympus BX41 microscopeandtheimageswerestoredforsubsequentanalysis.