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1 The Separation of Benign and Malignant Mesothelial Proliferations: Lessons from Morphology and Molecular Biology Andrew Churg, MD Departments of Pathology University of British Columbia & Vancouver General Hospital Vancouver, BC, Canada Notice of Faculty Disclosure In accordance with ACCME guidelines, any individual in a position to influence and/or control the content of this CME activity has disclosed all relevant financial relationships within the past 12 months with commercial interests that provide products and/or services related to the content of this CME activity. I do not have any conflicts of interest under ACCME/ASCP policy But I do consult for law firms in asbestos litigation Benign vs Malignant Mesothelial Proliferations Separation of pleural mesotheliomas from metastatic carcinomas Generally an issue of one very poorly treatable malignancy vs another Separation of mesothelioma from benign mesothelial reactions Difference in prognosis is vast Overcalling benign reactions tells patients they have an incurable malignancy 1

2 Separation of Benign vs Malignant Mesothelial Proliferations US-Canadian Mesothelioma Reference Panel Data Total circulated cases Percent of cases with disagreement about benign vs maligant 22% Do Clinical Features Help? Nodular pleural thickening strongly suggestive of malignancy Pleural thickening involving the mediastinal pleura strongly suggestive of malignancy Be cautious in diagnosing mesothelioma in the absence of pleural thickening on CT scan or direct visualization of tumor by operator An effusion by itself could be anything Causes of a Pleural Effusion in a Contemporary Population (Walker et al: Chest 2017) Etiology Number of Cases Percent of Cases Malignant % Cardiac failure 86 11% Hepatic failure % Renal failure % Pleural space infection % Misc inflammatory % Benign asbestos effusion % Post-CABG 6 0.7% Pulmonary embolism 5 0.7% Miscellaneous % 2

3 Distribution of Proliferating Mesothelial Cells and Malignancy In a Thickened Pleura Benign or malignant Layering (benign) Thick pleura Usually benign Malignant Fat 1 Stromal Invasion The best indicator of malignancy with mesothelial proliferations (when present!) Can sometimes be difficult to separate from entrapment Linear arrays of mesothelial cells tend to be entrapped Sharp circumscription of mesothelial proliferations (= lack of invasion) tends to be benign Pan-keratin staining very useful to show where the mesothelial cells are located But pan-keratin staining per se is not a marker of a benign or malignant process all active mesothelial proliferations are keratin positive Be cautious diagnosing invasion in the presence of a significant inflammatory infiltrate A solid nodule of tumor. Obviously malignant but not obviously invasive MP2375 3

4 Mesothelioma: full thickness spread in a thick pleura MP1974 Note relatively bland & monotonous cytology MP1974 A Subtle Peritoneal Mesothelioma 4

5 At Subtle first peritoneal glance all surface proliferation, mesothelioma no invasion MP2705 Very focal invasion: invasion of fat by mesothelial cells is always malignant Stromal Invasion The best indicator of malignancy with mesothelial proliferations (when present!) Can sometimes be difficult to separate from entrapment Linear arrays of mesothelial cells tend to be entrapped Sharp circumscription of mesothelial proliferations (= lack of invasion) tends to be benign Pan-keratin staining very useful to show where the mesothelial cells are located But pan-keratin staining per se is not a marker of a benign or malignant process all active mesothelial proliferations are keratin positive Be cautious diagnosing invasion in the presence of a significant inflammatory infiltrate 5

6 Linear Array Organizing Pericarditis MP2160 CK7 6

7 ? Invasion MP924 Not Invasion: En Face Cut MP924 Limit to Penetration vs Invasion MP1696 7

8 MP1696 MP1696 Keratin MP1696 8

9 Benign reaction Sharp circumscription Limit to penetration MP1696 Mesothelioma: Keratin MP584 Separating Invasion from Entrapment Be cautious diagnosing invasion in the presence of a major inflammatory reaction Linear arrays tend to be entrapped Sharply circumscribed mesothelial proliferations tend to be entrapped En face cuts are a major problem in small biopsies If in doubt it s better to report the process as atypical mesothelial hyperplasia 9

10 Criteria for the Diagnosis of Desmoplastic Mesothelioma Bulk of lesion is paucicellular and shows a storiform or patternless pattern plus Stromal invasion or Bland necrosis or Overtly sarcomatous foci or Nodular stromal expansions or Distant metastases Desmoplastic mesothelioma MP2091 Desmoplastic mesothelioma: patternless pattern of Stout + ropey collagen MP

11 Desmoplastic mesothelioma MP2091 Keratin: invasion away from pleural surface MP2091 Keratin Invasion of fat

12 Bland Necrosis Pan-keratin in area of bland necrosis MP2320 Nodular Stromal Expansion Indicates tumor is producing its own stroma Can be viewed as variant of stromal invasion Usually indicator of malignancy 12

13 Desmoplastic mesothelioma Nodular stromal expansion MP790 Features of Organizing Pleuritis * Cellularity greatest immediately under effusion Progressively fibrotic and paucicellular away from effusion ( zonation ) Capillaries perpendicular to surface Cells immediately under effusion can be very atypical, particularly when mesothelial cells are mixed with fibrin Examine areas away from fibrin All active mesothelial proliferations are keratin + Fibrosis can extend into fat (but keratin negative)! * Also called fibrosing pleurisy, organizing pleurisy Organizing pleuritis MP918 13

14 MP918 Organizing pleuritis MP1045 Organizing pleuritis with marked surface atypia MP

15 Organizing pleuritis (organizing empyema) VS Organizing pleuritis (organizing empyema) VS

16 Keratin: sharply demarcated line of positivity no keratin positive cells in fat Keratin (hydrocoele): lamellar arrays MP1147 Ancillary Techniques NB: Ancillary techniques are ancillary. First look at the morphology! Traditional immunohistochemical markers Markers based on genomic alterations 16

17 Churg and Galateau-Salle Arch Path 2012 Keratin Staining In Mesothelial Proliferations All active mesothelial proliferations are keratin positive Benign proliferations Malignant proliferations Keratin staining does not make a mesothelial proliferation malignant Keratin staining tells you where the mesothelial cells are located Logic Behind Proposed IHC Markers Empiric Markers EMA-claimed to be a marker of malignancy in this context Claims are purely empiric no known mechanism(s) or molecular observations in support Desmin-claimed to be a marker of benignancy. Claims are purely empiric no known mechanism(s) or molecular observations in support Not useful!!!! 17

18 Logic Behind Proposed IHC Markers Markers with some notional mechanistic or molecular basis Glut-1: Many tumors are hypermetabolic Glut-1 stains red cells and staining very hard to interpret Some benign reactions stain with Glut-1 IMP-3: Expressed in many malignant tumors Not known if IMP-3 is abnormal in mesotheliomas Some benign reactions stain with IMP-3 p53: p53 is mutated in many tumors. However, p53 is usually not mutated in mesotheliomas What has been called p53 staining is probably mostly wild type Mutational Landscape (Tumor Suppressor Genes) of Mesothelioma Yap et al: Nat Rev Can 2017 Wild type p53 in a mesothelioma MP

19 % Surviving Serous Carcinoma P53 null Serous Carcinoma True Mutated p53 Patterns P53 100% staining VC Survival at 5 Years by IHC Stain Result (Cutoff: 10% Staining) (Churg and Galateau-Salle, Arch Path 2012) Survival at 5 Years Using a 10% Staining Cutoff Desmin EMA p Desmin <10% Desmin >10% EMA <10% EMA >10% p53 <10% p53 >10% 16 Cancer Cytopath 2003 [CDKN2A = p16 INK4a ] Clin Can Res 2012 Can Res

20 Function & Detection of Genes Frequency Mutated or Deleted in Mesotheliomas p16 INK4a (CDKN2A): Prevents cell cycle progression. Tumor suppressor. Loss of p16 (9p21 locus) can be detected by FISH p16 IHC does not give the same information as p16 FISH! Reactive conditions can have p16 methylation and hence no staining When p16 is expressed in mesotheliomas, the expression is patchy BAP1: controls DNA repair and genes related to cell proliferation, cell cycle, cell death. Believed to function as a tumor suppressor. Wild type BAP1 protein is detectable by IHC Deletions or mutations in BAP1 lead to loss of IHC-detectable nuclear protein (protein either lost or sequestered in cytoplasm) NF2: Regulates cell proliferation in response to adhesion. Functions as a tumor suppressor Detection of abnormal NF2 in tissue sections problematic Chromosome 9 From Abbott/Vysis Site 20

21 Benign Reaction Mesothelioma Chromosome 9 centromere-- green p16 probe--red Fraction of Benign Mesothelial Reactions Showing Homozygous Deletion of p16 by FISH Report Number Percent Illei /19 0% Chiosea /40 0% Monaco /70 0% Chung /11 0% Wu /10 0% Sheffield /40 0% Ito /30 0% Total 0/220 0% Churg et al: Arch Path 2016 Epithelial Mesotheliomas Showing Homozygous Deletion of p16 by FISH Report Pleural Peritoneal Chiosea % 25% Monaco % 51% Kasinskas % Chung % Takeda % Wu % Hwang % 14% Sheffield % Ito % Singhi % Churg et al: Arch Path

22 Mixed/Sarcomatous Mesotheliomas Showing Homozygous Deletion of p16 by FISH Report Morphology % Deleted Chung 2010 Mixed/Sarcomatous 33% Takeda 2010 Mixed 86% Takeda 2010 Sarcomatous 100% Wu 2013 Sarcomatous 100% Tochigi 2013 Sarcomatous 81% Sheffield 2015 Sarcomatous 50% Hwang 2016 Sarc/desmoplastic 80% Singhi (peri) Mixed/sarcomatous 27%? Desmoplastic meso * MP2077 p16 FISH 22

23 Bx suspicious for mesothelioma MP2380 Cautions re p16 FISH for Diagnosing Mesothelioma vs Reactive Proliferation Only loss of p16 is diagnostic Losses must be above the truncation background Reasonably sensitive test for sarcomatous mesotheliomas Roughly 30% of epithelial pleural mesotheliomas and at least 50% of epithelial peritoneal mesotheliomas do not show homozygous p16 deletion p16 IHC cannot be substituted for p16 FISH: p16 IHC does not give the same information p16 loss can be seen in many types of malignancies, so first be sure you are dealing with a mesothelial proliferation Run BAP1 IHC first if you have it it s much cheaper and faster than p16 FISH 23

24 Function & Detection of Genes Frequency Mutated or Deleted in Mesotheliomas p16 INK4a (CDKN2A): Prevents cell cycle progression. Tumor suppressor. Loss of p16 (9p21 locus) can be detected by FISH p16 IHC does not give the same information as p16 FISH! Reactive conditions can have p16 methylation and hence no staining When p16 is expressed in mesotheliomas, the expression is patchy BAP1: controls DNA repair and genes related to cell proliferation, cell cycle, cell death. Believed to function as a tumor suppressor. Wild type BAP1 protein is detectable by IHC Deletions or mutations in BAP1 lead to loss of IHC-detectable nuclear protein (protein either lost or sequestered in cytoplasm) NF2: Regulates cell proliferation in response to adhesion. Functions as a tumor suppressor Detection of abnormal NF2 in tissue sections problematic BAP1 lost mesothelioma. Arrows indicate positive stromal cells and lymphocytes 24

25 BAP1 positive mesothelioma. Arrows indicate positive stromal cells and lymphocytes Frequency of BAP1 Loss by IHC in Benign Reactions (Tissue) Sheffield AJSP 2015 & Churg unpublished 0/53 Galateau-Salle unpublished 0/23 Cigognetti Mod Path /25 McGregor Human Path /20 Churg et al: Arch Path 2016 Frequency of BAP1 Loss by IHC in Mesotheliomas Report N Epi Mixed Sarc Nasu % 59% 50% Farzin % 42% 18% Yoshikawa % %---- Sheffield 2015* 22 56% 10% Cigognetti % 60% 15% Hwang % Singhi 2015** 75 64% %--- McGregor % 0% Righi 2016* % 22% Tandon 2017** % *pleural ** peritoneal Modified from Churg et al: Arch Path

26 Loss of BAP1 in the epithelial but not sarcomatous component of a biphasic mesothelioma Core bx from patient with nodular pleural thickening (cells mark as mesothelial) MP

27 BAP1 Cautions Re BAP1 IHC Loss of staining is diagnostic of mesothelioma; however Only nuclear staining is of interest A significant proportion (30 to 40%) of epithelial mesotheliomas will not show BAP1 loss Most sarcomatous/desmoplastic mesotheliomas will not show BAP1 loss Crucial that sections show a positive internal control (inflammatory or stromal cells) Other tumors (ocular & cutaneous melanomas, some renal cell ca, others) can have BAP1 loss in the BAP1 cancer syndrome and as occasional somatic mutations Confirm that process is mesothelial before using BAP1 Human Pathology 2016 BAP1 loss found in 1% of non-small cell lung carcinomas 27

28 Andrici et al: Human Path 2016 BAP1 loss found in <1% of high grade serous carcinomas Function & Detection of Genes Frequency Mutated or Deleted in Mesotheliomas p16 INK4a (CDKN2A): Prevents cell cycle progression. Tumor suppressor. Loss of p16 (9p21 locus) can be detected by FISH p16 IHC does not give the same information as p16 FISH! Reactive conditions can have p16 methylation and hence no staining When p16 is expressed in mesotheliomas, the expression is patchy BAP1: controls DNA repair and genes related to cell proliferation, cell cycle, cell death. Believed to function as a tumor suppressor. Wild type BAP1 protein is detectable by IHC Deletions or mutations in BAP1 lead to loss of IHC-detectable nuclear protein (protein either lost or sequestered in cytoplasm) NF2: Regulates cell proliferation in response to adhesion. Functions as a tumor suppressor Detection of abnormal NF2 in tissue sections problematic Mod Pathol 2016 In peritoneal mesotheliomas, homozygous loss of p16 or hemizygous loss of NF2 by FISH was associated with a poor prognosis. BAP1 loss did not affect prognosis. 28

29 p16 not lost p16 lost NF2 not lost NF2 lost The Pure Surface Proliferation Problem Since invasion is the most useful diagnostic clue to malignancy in mesothelial proliferations, what to do with pure surface processes? A. If there is radiologic or visual evidence of tumor B. If the pleura/peritoneum is radiologically and visually normal (ie, how do we recognize mesothelioma in situ?) Male w pleural tumor on imaging. No invasion seen in biopsy MP

30 BAP1 BAP1 Conclusion: malignant mesothelioma 30

31 The Pure Surface Proliferation Problem Since invasion is the most useful diagnostic clue to malignancy in mesothelial proliferations, what to do with pure surface processes? A. If there is radiologic or visual evidence of tumor B. If the pleura/peritoneum is radiologically and visually normal (ie, how do we recognize mesothelioma in situ?) The Concept of Mesothelioma in Situ D Whitaker, DW Henderson, KB Shilkin Sem Diag Pathol 1992; 9: Seven cases All had areas of invasive mesothelioma In situ mesothelioma = single or multiple layers of surface cells 1?Mesothelioma in Situ?

32 Reaction to Pneumothorax Presumed MIS + Mesothelioma 1 2 MP1342 MP896 3 Presumed Mesothelioma in Situ + Mesothelioma 4 MP1497 PHR ?Mesothelioma in Situ?

33 Consensus Opinion Mesothelioma in situ cannot be reliably recognized by routine morphology Consensus Opinion Mesothelioma in situ cannot be reliably recognized by routine morphology What about molecular markers? Two Examples of True Mesothelioma in Situ Case 1: 70 F presented with cough, found to have a large pleural effusion which was drained but recurred x 4 Imaging showed only an effusion 1 year after initial presentation underwent thoracoscopy: pleura reported as thin and smooth Biopsy performed and reported as benign 33

34 Case 1 First bx MP2662 BAP1 p16 Two Examples of True Mesothelioma in Situ Case 1 continued: Over the subsequent 9 months again had recurrent effusions A second thoracoscopy performed: pleura again thin and smooth A second biopsy performed Case 1 2 nd Bx BAP1 p16 NF2 34

35 Case 1: Summary of Morphologic Findings First biopsy Single layer of bland mesothelial cells BAP1 lost p16 FISH: homozygous deletion in 3.8% of cells and hemizygous deletion in 22.5% of cells (exceeds bkg range) Second biopsy Single layer of bland mesothelial cells BAP1 lost p16 FISH: homozygous loss in 10% of cells and hemizygous loss in 23.0% of cells (exceeds bkg range) NF2 FISH did not show hemizygous or homozygous loss, but hyperploidy was present in 28% of cells Two Examples of True Mesothelioma in Situ Case 1 continued: Over the subsequent 6 months she continues to have pleural effusions There is no radiologic evidence of tumor Now 25 months from initial presentation CT Scan 25 Months After Initial Presentation 35

36 Case 2 60 F, past history breast ca, presented with abdominal bloating Imaging showed ascites and a suggestion of minimal omental thickening At laparoscopy there was extensive ascites, the omentum looked very slightly thickened but not malignant, and the peritoneal surfaces otherwise appeared grossly normal Omental biopsy performed and initially interpreted as a reactive mesothelial hyperplasia Developed recurrent ascites which was tapped multiple times Tentative diagnosis of idiopathic sclerosing peritonitis was made Treated with Tamoxifen. Died 27 months after presentation Case 2: Presentation Case 2 BAP1 Omental biopsy showed rare tiny foci of superficial invasion, but mostly a single layer of mildly atypical mesothelial cells 36

37 p16 NF2 Case 2: Summary of Morphologic Findings Omental biopsy Single layer of mildy atypical mesothelial cells Rare tiny foci of superficial invasion BAP1 lost p16 FISH: homozygous loss in 10% of cells and hemizygous loss in 30% of cells (normal range 0 to 20%) NF2 FISH: homozygous loss in 3.6% of cells and hemizygous loss in 3.6% of cells (normal range 0 to 20%). 6.0% of cells demonstrated hyperploidy Conclusions Mesothelioma in situ as defined by in situ molecular testing does exist These cases are not recognizable by routine morphology Whether this process always becomes invasive and how long that takes remains to be determined If you are suspicious but molecular markers fail, do not diagnose mesothelioma in situ! Call it atypical hyperplasia 37

38 Separation of Benign from Malignant Findings in Mesothelial Cells Finding Significance Stromal invasion Always malignant Linear arrays Favors benign process Nodular stromal Usually malignant expansion Zonation Usually benign process Necrosis Usually but not always malignant Cytologic atypia Often misleading Pure surface Problematic, occasionally diagnosable proliferation with BAP1 or p16 FISH. Immunochemistry BAP1 loss useful, except for keratin, everything else is not! Molecular testing p16 FISH homozygous deletion useful NF2 FISH hemizygous deletion useful 38