Recent advances in cytolical diagnosis of mesothelioma

1. Cytological Diagnosis of Mesothelioma :Recent
Advances
Dr.NEHA SINGH
Dr.MUKUL SINGH
Dr.NAVEENVMMC & SAFDARJUNG HOSPITAL,NEW DELHI

2. Contents
 Introduction
 Etiology/Risk factors
 Pathogenesis
 Cytological Diagnostic Approach
 Biomarkers
 Prognostic Parameters
 Recent advances in Treatment
Modalities

3. Introduction


At that time, the diagnosis was extremely
controversial
(required autopsy examination)
Today, the diagnosis is still problematic
– 15 % of cases cannot be differentiated
from Adenocarcinoma
The term was first used in 1921 by
Eastwood & Martin
to describe primary tumors of the pleura

4. Incidence
• Industrialized Countries
– 2 per million in females
– 10 - 30 per million in males
Regional differences are due to the level of industrial activity
 Areas with shipyards are at the highest
risk

5. The occurrence of mesothelioma is mostly related
to an
Occupational Exposure to Asbestos


Only 7.2 % of asbestos workers, will develop the disease
Up to 50 % of patients, do not have any history of exposure

6. Risk Factors

7. Uses of Asbestos
Friction materials
(brakes, clutches, etc.)
Asphalt and vinyl felts
Papers and adhesives
Flooring and roofing
materials
Filters, sealants and
gaskets
Vinyl flooring
Sprayed-on fireproofing material

8. Types of Asbestos
 serpentine
fibres
Chrysotile “White
Asbestos”
 Amphiboles fibres
Amosite “Brown
Asbestos”
Crocidolite(Dangerous)
Tremolite
Chrysotile fibers,
high magnification
Most
common:
Others Types:
mostly found as
contaminants
in other materials
 Actinolite
 Anthophyllite

9. • Type of Fibers:
Amphibole > Chrysotile
Crocidolite and Amosite – the main cause of mesothelium
• Fiber Length
Longer fibers (5-20 µm)
• Naturally Occuring Carcinogenic Mineral Fibers:
Erionite - <5µm
Most potent fiber in causing Mesothelioma
Antigorite
Chrysolite
Tremolite
• In case of environmental Exposer:
M:F = 1:1
Mesothelioma tends to occur at younger age (<55yrs)
Strength of Association with Malignant Mesothelioma

10. Simian Virus 40 Exposure
polio vaccine contaminant in the
50s & 60s – On review by
International Agency for
research on Cancer – Evidence
was inadequate

11.  Recently, interest regarding the hypothesis that Talc, and/or
talc contaiminated with asbestos cause Mesothelioma and
other cancers. – Not any supporting mechanistic studies or of
experimental studies.
 However, use of perineal Talc – classified as possible human
Carcinogen.
Exposure to Talc

12.  Radiation therapy
• Radiation to supradiaphragmatic fields
• Intrapleural thorium dioxide
Zeolites
• microporous,aluminosilicate minerals , used as catalysts
• An example is erionite which is common in Turkey
Other Potential Causes :

13. Carcinogenic Mechanism
On inhalation, carcinogenic mineral fibers reaches lung parenchyma
Through lymphatics, reaches pleura and peritonium,remains for months or yrs
Triggers Chronic inflammation driven by High mobility group protein B1(HMGB1)
secretion
Induces NF-kB and PI3K pathways
Favors proliferation of spontaneously mutated mesothelial cells
Mesothelioma

14. Role of Genetics
Current concept – GxE interaction
G – Inherited/Germline BAP1 mutation
E – Environmental exposure to asbestos/carcinogenic fibers
Inherited BAP1 mutation – ‘BAP1 cancer syndrome”
Uveal melanoma
skin melanoma
RCC of clear cell variant
breast cancer
cholangiocarcinomas
various types of brain tumors

15. BAP1
 BAP1-BRCA1 Associated Protein-1
 Itisadeubiquitylase–regulatesDNAreplication,DNArepair,Metabolismandcelldeath.
 BAP1gene is located on chromosome 3

16. In cytoplasm,it modulates the stability of
IP3R3 channel
Allows flux of Ca2+ from Endoplasmic
Reticulum into mitochondria(Krebs cycle)
At higher doses – execute apoptosis

17.  Additional Germline Mutations:
MLH1
MLH3
TP53
BRCA 2
CDKN2A
NF2
LATS2
SETD2

18. Newer Studies
 IN 2016,Yoshikawa et al. – Discovered that Chromothripsis (chromosome
shattering followed by random chromosomal rearrangements) – causes
genetic alteration in mesothelioma
Single large catastrophic
event
Abberant repair

19.  Another recent theory proposed by Mansfiel et al. - predicted that vast array of
genetic alterations in mesothelioma – may lead to production of Neoantigens ,
which correlated with the clonal expansion of tumor – infiltrating T lymphocytes.
These newer concepts suggest that Mesothelioma may be
immunogenic. Hence, Future targeted deep-sequencing studies and
single-cell analyses will provide further insights in the pathogenesis
of Mesothelioma

20. WHO, 2015

21. Malignant Mesothelioma
• 3 Cell Types
– Epithelial Type : 50 % of cases
• most often confused with adenocarcinoma
– Mesenchymal Type : 16 % of cases
– Mixed Type : 34 % of cases

22.  As 90% of patients present with a serous effusion, regardless of histologic
subtype (epithelioid, mixed, and sarcomatoid) , effusion sample are the
first to be examined in the diagnostic work-up of patients with MPM.
 Pleural samples can be divided into three groups (a/c IAC and IMIG):
a. malignant effusions on cyto-architectural grounds
b. effusions that require some form of ancillary testing to establish malignancy
c. non-diagnostic effusions (minimal cell shedding, i.e., almost all sarcomatoid
MPMs, and those epithelioid MPMs that do not have malignant
cytomorphology, and that are negative for ancillary tests of malignancy).

23.  When effusion cytology is inconclusive for the diagnosis, tissue biopsies
should be taken.
 Once diagnosis is made, prognostic stratification and also predictive tools
should be defined.
 Nowadays, the main prognostic parameter is still represented by the
histological subtype, having the epithelioid MPM a better outcome than the
sarcomatoid or biphasic MPM.
 Reliable predictive biomarkers are still lacking in MPM and a personalized
therapeutic concept is eagerly needed.

24.  The main obstacles for the diagnosis are :-
(i) low content of diagnostic cells due to bleeding or inflammation, or
recurrent effusions and repeated thoracocentesis.
(ii) when the tumor is a sarcomatoid MPM or it is dominated by a
sarcomatoid component (mixed MPM)
(iii) lacking experience of the cytopathologist and unawareness of this
diagnostic possibility.

25. Approach to cytological Diagnosis

26. Cytomorphology of Malignant
Mesothelioma
Figure : Cytomorphology of epithelioid mesothelioma cells and tissue fragments in effusions.
The tissue fragments sometimes show gaps or windows, (a and b) occur both as spheres with
smooth surfaces (c and d) and in berry-like clusters with scalloped surface (e and f). The tissue
fragments may contain acidophilic extracellular matrix cores also known as collagen or basement
membrane cores (g and h) which with May-Grünewald-Giemsa (MGG) becomes strongly
acidophilic, similar to the extracellular granular material that can be seen in the background
[Figures 1 and 2f], indicating large amounts of hyaluronan (left PAP stain, right MGG stain; bar =
50 μm). Photo: VA frames a, c, e and g; AH frames b, d, f and h

27.  Mesothelioma cells are significantly larger (5-10 times) than
normal mesothelial cells. Nuclei are centrally located, are
usually bland but show very prominent eosinophilic
nucleoli (Fig. A).
 Cells may be bi-nucleated (Fig. B) or multinucleated (Fig.
C). The cytoplasm is dense and abundant.
 In Pap-stained smears, mesothelioma cells show different
staining in central and peripheral cytoplasm (a two-tone
staining) and the “blebbing” with cytoplasmic protrusions at
the plasma membrane (Fig.B).
 The cytoplasm often contains very small vacuoles that
represent ethanol-extracted lipids that are best seen in MGG
stained smears (Fig. D).
 A suggestive finding that establishes the mesothelial origin
is the occurrence of intercellular slits or ‘windows’ and cell-
engulfment (Fig. E).

28.  The two main differential diagnosis to MPM are :
• Metastatic Adenocarcinoma
• benign, reactive mesothelial cells.

29. Features Malignant
Mesothelioma
Metastatic
Adenocarcinoma
Cell arrangements Monotonously
arranged
Haphazardly
arranged
Cell clusters Peripheral collar of
cytoplam
Absent
Nucleus location Centrally placed Show polarity
Nuclear Atypia Relatively less Marked
Nuclear Irregularity Relatively less Marked
Nucleoli Prominent Prominent
Multinucleation Less common Frequently present
Metastatic Adenocarcinoma

30. Features Malignant
Mesotheliom
a
Mesotheliosis
Arrangement 3-d ball Monolayers of flattened
mesothelial cells
Mitosis Less, but
atypical
mitosis
favours MM
plentiful
Necrosis Markedly
present
Absent
Vacuoles If present ,
large
vacuoles.
May be present but they
don’t occupy the entire
cell(small vacuoles)
Lack targetoid vacuoles
Reactive Mesothelial cells
Atypical mesothelial cell forming 3-d cluster is also noted
in some benign cases.

31. Immunocytochemistry and
Immunohistochemistry
 The main differential diagnoses of MM are:
• adenocarcinoma
• reactive mesothelial proliferations, so called “mesotheliosis,”
 It can be resolved using ICC on cytospins or IHC on cell blocks. Ancillary
testing must be available in laboratories diagnosing MM based on
effusions.

34. - No specific panel of antibodies recommended(depends
on antibody clone and laboratory experience)
- Antibodies chosen should have a sensitivity and
specificity of at least 80%
- TWO MESOTHELIAL AND TWO EPITHELIALMARKERS

37. Malignant mesothelioma versus reactive mesothelium
(“mesotheliosis”)
 It is more difficult to distinguish reactive mesothelial cells from malignant
ones based on IHC or ICC and at least two antibodies should be used in a
panel.
 Benign mesothelial cells are known to contain muscle filaments and they
express desmin.
 The mesothelial cell loses this reactivity to desmin early during the
oncogenic process, and the absence of such immunoreactivity is a
commonly recommended criterion for MM.
 In the same way, the typical cell membrane reactivity to EMA in the
mesothelial cell is a strong indicator of malignancy,

38. Reactive versus neoplastic?
Immunohistochemical stain Reactive Mesothelioma
Desmin Positive Mostly negative
EMA Negative Positive
IMP3 Negative Positive
GLUT1 Negative Positive
P53 Negative Positive

39. Peritoneal and pericardial malignant
mesothelioma
 The challenging differential diagnosis in the peritoneum is between MM and
ovarian or peritoneal serous carcinoma.
 the serous carcinomas of ovary and uterus and peritoneal origin stain positive for
BerEp4, PAX8, B72.3, BG8, CA 19-9, Leu-M1 (CD15), MUC4 and estrogen
receptor, all of which are rarely or never expressed in MM.
 Pericardial MM are less common.
 The diagnosis is based on the same criteria as pleural and peritoneal MM, but
pericardial fluids are often associated with inflammatory conditions, and here the
challenge is to distinguish the MM from mesothelial reactive changes.
 ICC and IHC along with FISH plays an important role in establishing diagnosis.

40. Epithelial or
mesothelial?
Do IHC’s (at least
two mesothelial
and twoepithelial
markers)
Mesothelial
Benign vs.
malignant?
Check features
todifferentiate.
Benign
Atypical
mesothelial
proliferation
Correlate with
clinical and
imagingfindings.
May test forBAP-
1 loss by IHC and
homozygous
deletion by p16
FISH.
Malignant
Mesothelioma
Epithelial
Markers for
differentiation
(TTF-1, p40,
GATA3,Pax-8,
etc.)
Summarized approach for cases with a
thickened pleura and/ or an atypical
proliferation within the pleura.

41. Recent Markers
 glucose transporter 1 (GLUT-1)
 insulin-like growth factor II messenger RNA-binding protein 3 (IMP-3)
They have been reported to have utility in differentiating benign and maligant
mesothelial proliferations. GLUT-1 and IMP-3 show positivity in mesothelioma
and negativity in reactive process.

42.  D2-40 – differentiate between epitheloid and sarcomatoid mesothelium.
+ve – Sarcomatoid
-ve – Epitheloid
Very sensitive but it lacks specificity
 Cam5.2 :
-ve – Epitheloid
+ve – Sarcomatoid (100%)
also +ve in metastatic sarcomatoid carcinoma , carcinosarcoma

43.  GATA3 –
+ve – MM
-ve – sarcomatoid carcinoma , except for urothelial tumor (GATA3 +ve)

44.  BAP1 protein
• detected by immunohistochemistry or by FISH,
• frequently lost in a large proportion of epithelioid or biphasic
mesotheliomas with a sensitivity higher than 60%.
• In this contest, the use of BAP1 immunostain can be useful in the
differential dignosis between benign and malignant mesothelial
proliferations.
• However, Its role in differential diagnosis between fibrous pleuritis and
sarcomatoid mesothelioma is controversial - in which BAP1 loss has been
observed in less than 20% of cases

45. Features of BAP1 ICC/IHC
 Routinely used in western countries
 Widely used – BAP1 (wild type)
 Normally, this protein is found in nucleus as well as cytoplasm , resulting in
• Strong nuclear staining
• Less intense cytoplasmic staining

46.  In case of BAP1 mutation – mutations occur in catalytic domain
Prevents the autodeubiquitylation of BAP1 which is required for it to enter the
nucleus.
 In case of BAP1 deletion – cause truncated BAP1 protein which lack
carboxy terminus that contains the nuclear localizations signal.
Hence in either of the cases , nuclear staining of BAP1 is completely
absent in MM
OR
Cytoplasmic staining can be present but without nuclear staining.

47.  So, benign cells always show nuclear positivity for BAP1.
 Overall ,approximately 70% of epithelial and 50% of sarcomatoid
mesothelioma shows negative nuclear stainig.
 However , BAP1 positive nuclear staining also present in 30-40% in
mesothelioma.
Diagnostic Dilemma

48.  In that cases, FISH analysis for p16 mutation to be done to establish
diagnosis

49. FISH technique
 A recent study has demonstrated that a proportion of mesotheliomas
exhibit also
• loss of neurofibromin 2 (NF2)
• large tumor suppressor kinase 2 (LATS1/2)
 Both detected by FISH that should be useful in differentiating benign form
malignant lesions of the pleura.
 This has not been confirmed by immunohistochemistry studies

50. Electron Microscopy
 EM can be of help when ICC and IHC are not entirely conclusive or when
the biomarkers indicate a MM in spite of inconclusive cytomorphology
 Effusions are well suited for EM, provided a portion of the cell pellet is
properly fixed early in the process. This requires that an aliquot of the first
fresh cell pellet is fixed in glutaraldehyde without delay.
 EM is considered the gold standard for the diagnosis of MM

51.  The finding of apical cell membranes
with microvilli at basolateral surfaces,
forming “extracellular neolumina,” and/or
forming an intracellular vesicle or
“intracellular neolumina” is
pathognomonic for a malignant cell
[Figure c and d].
 To be certain that the group of cells
represent a tissue fragment of the tumor
and not just an aggregate of cells,
desmosomes should be present between
the cells [Figure d]
Figure : Electron microscopy of malignant mesothelioma (MM) in effusions. The
nuclear pleomorphism is normally more extensive than seen in cytomorphology: (a and
b) Typically, the apical surface of MM cells is covered by long, slender microvilli,
completely devoid of any glycocalyx, and malignancy is shown by the finding of
neolumina, that is, apical cell membranes with microvilli as either a + cytoplasmic
vacuole (c) or in-between cells with connecting desmosomes, replacing the basolateral
membrane. (d) Cytoplasmic filaments are often seen surrounding the nucleus as a
“scarf”, (e) sometimes forming coarser tonofibril structures [f; also cf. Figure 3 g]

52. Genetic Testing
 Although MM is considered as a malignany that cannot be cured
surgically, early diagnosis is a/w better response to therapy and survival of
10 yrs or more.
 Genetic testing of patients with familial history of mesothelioma or other
cancers at a younger age (<50 yrs) by NGS.
 Ideally, all patients with mesothelioma should undergo genetic testing
along with genetic counseling (because of GxE theory)

53. Biomarkers
 Serum mesothelin:
• Most widely used biomarker
• Its level increases in MPM as compared to benign pleural disease.
• Soluble mesothelin-related peptides (SMRP) in pleural fluid have high
specificity (95%) but has sub-optimal sensitivity (32%)
• It is negative in both sarcomatoid and in half of epitheloid
subtypes,especially in the early stages.
• Plays an important role in monitoring treatment response as SMRP level
correlates with tumor size and progression

54.  Osteopontin
• A glycoprotein – overexpressed in several malignancies , including MPM.
• A study by Pass et al. found found that OP levels in tumour tissue, but not
serum, were significantly elevated in MPM when compared to healthy
controls with and without asbestos exposure.
• both serum and plasma OP levels correlated with survival.
• Elevated OP levels have also been associated with other malignancies,
causing a low diagnostic specificity
Ref: Pass HI et al. Asbestos exposure, pleural mesothelioma, and serum osteopontin levels. N Engl J Med. 2005;353(15):1564-73.

55.  Hyaluronan (HA):
• a polysaccharide expressed in high levels in the serum and pleural fluid of patients with
MPM, is another established biomarker.
• Pleural fluid levels of >100,000 ng/mL have been recommended as a diagnostic
indicator for MPM.
• elevated intracellular HA levels have also been associated with MPM.
• ability to differentiate between MPM and metastatic adenocarcinoma, since
mesothelioma cells express high levels of intracellular HA, a feature not found in
metastatic adenocarcinoma.
• Serum HA levels are higher in patients with later or progressive stages compared to
responders, suggesting that HA is a marker of progressive disease.28

56.  MicroRNA (miRNA):
• are short, non-protein coding single-stranded RNA involved in the regulation of gene
expression and can contribute to either oncogenesis or tumour suppression.
• different miRNA expression profiles in MPM tissues, serum, and pleural fluid can be studied
using microarray profiling and quantitative real-time polymerase chain reaction.
• miR-126 has been consistently shown to be downregulated in MPM tissue compared to
normal pleuraI. It has high sensitivity but specificity is low
• Plasma miRNA, such as miR-625-3p have also been validated as diagnostic markers for MPM.

57. Prognostic Biomarkers

58.  A recent study by Ujiie et al (1), have shown that:
• Tumor with high CD163 – positive tumor associated macrophages and low CD8
positive T lymphocyte infilteration – Worst prognosis
• Patient with low CD163 positive tumor associated macrophages and high CD8
positive T lymphocyte infilteration – Good prognosis.
 A new Study by Mansfield et al.(2) have shown that :
• 39% of patients show PD-L1 expression and is associated with worse prognosis.
• Its expression was higher in non-epitheloid mesotheliomas.
Ref 1: Ujiie H, Kadota K, Nitadori JI, et al. The tumoral and stromal immune microenvironment in malignant pleural mesothelioma: a comprehensive analysis reveals prognostic immune markers. Oncoimmunology.
2015;4:e1009285.
Ref 2:Mansfield AS, Roden AC, Peikert T, et al. B7-H1 expression in malignant pleural mesothelioma is associated with sarcomatoid histology and poor prognosis. J Thorac Oncol. 2014;9:1036-1040.

59. Recent Advances in Therapy
Standard 1st line regimen pemetrexed pluscisplatin or carboplatin
Improvessurvival, but only by a few monthson average
Improvesquality of life
Recently , the DREAM (Durvalumab with First-line Chemotherapy in Mesothelioma) as a first
line therapy is being investigated.
• Durvalumab – PD-L1 inhibitor
• Regime – cisplatin and pemetrexed – 6 cycles
followed by maintainance durvalumab – every 3 wks
• Its in Phase 3 trial.

60. BAP1 as a Therapeutic Target
• BAP1 loss causes upregulation of EZH2 (methyltransferase) which causes in hypermethylation of histone
H3 causing chromatin modulation and hence leading to MM
Current trial going on EZH2 inhibitor - Tazemetostat– Phase 2 trial
• A study by Mutti et al. have shown that patients are more sensitive to chemotherapy (gemcitabine) with
functional BAP1 compared with cells bearing mutated BAP1.
• Cells with BAP1 mutation – more sensitive to radiotherapy and t/t with Olaparib – a PARP inhibitor.

61. Ongoing Clinical Trials
Drug Line Phase Sponsor
Bevacizumab 1st with pem/cis II/III French Intergroup
Cediranib 1st with pem/cis RP II SWOG
NGR-hTNF 2nd line RP II MolMed
Pemetrexed Maintenance RP II Alliance
WT-1 vaccine Adjuvant RP II MSKCC, MD
Anderson

62. Potential FutureClinical Trials
Drug Sponsor
BAY 94-9343 Bayer
GDC-0980 Genentech
MORAb 009 Morphotek
Nintedanib Boehringer
Ingelheim
Tremelimumab MedImmune
VS-6063 Verastem

63. Thank you !