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Treatment of patients with acute lymphoblastic leukemia with bulky extramedullary disease and T-cell phenotype or other poor prognostic features Randomized controlled trial from the Children's Cancer Group

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583
The Use of Epithelial Membrane Antigen and SilverStained Nucleolar Organizer Regions Testing in the
Differential Diagnosis of Mesothelioma from Benign
Reactive Mesothelioses
Karen D. Wolanski, B.Sc. (WAIPMR. WA.)
Darrel Whitaker, Ph.D. (WAIPMR. WA.)
Keith B. Shilkin, M.B.B.S. (WAIPMR. WA.)
Douglas W. Henderson, M.B.B.S. (FMC.
SA.)
Western Australian Centre for Pathology & Medical Research, Queen Elizabeth II Medical Centre, Nedlands, Australia; Flinders Medical Centre, South Australia.
BACKGROUND. The accurate diagnosis of pleural lesions obtained from small closed
biopsy is difficult. As yet there is no single reliable test to distinguish between
malignant and benign mesothelial tissue.
METHODS. Immunostaining of epithelial membrane antigen (EMA) and the quantitation of silver stained nucleolar organizer regions (AgNORs) each were applied
to benign and malignant histologic sections of pleural and peritoneal biopsies.
The usefulness of these stains was tested both individually and in combination in
the diagnosis of epithelial malignant mesothelioma.
RESULTS. One hundred and three of the 141 malignant lesions (73%) were immunoreactive for EMA but only 3 of the 73 benign lesions (4%) reacted equivocally, and
none positively. The average count of AgNORs/cell in malignant lesions (n Å 80)
was elevated compared with benign cases (n Å 26), but a significant overlap was
exhibited in the AgNOR count and this form of analysis was considered to be of
little value in distinguishing benign from malignant mesothelial processes. Much
less overlap was observed when the average AgNOR area was measured. By using
the maximum benign AgNOR area of 0.6677 mm2 as the upper threshold, 51 cases
(63.8%) were identified as malignant; the test demonstrated 100% specificity and
63.8% sensitivity. By combining the EMA and AgNOR results, 76 of 80 of the malignant mesothelioma cases (95%) tested positive for at least 1 of the tests with no
false-positive results identified.
CONCLUSIONS. This study confirms the usefulness of EMA in diagnosing malignant
and benign mesothelial lesions, and demonstrates the enhanced diagnostic value
of combining EMA immunoreaction with the average area of AgNOR per cell,
thereby increasing sensitivity in the diagnosis of epithelial malignant mesothelioma. Cancer 1998;82:583–90. q 1998 American Cancer Society.
KEYWORDS: diagnosis, pleural biopsy, mesothelioma, epithelial membrane antigen,
silver-stained nucleolar organizer regions.
Support for this work was provided by Sir
Charles Gairdner Hospital Research Foundation
and the State Government Insurance Commission of Western Australia.
Address for reprints: Darrel Whitaker, Ph.D.,
Western Australian Centre for Pathology and
Medical Research, Queen Elizabeth II Medical
Centre, Locked Bag 2009, Nedlands 6009, Australia.
Received February 10, 1997; revision received
June 19, 1997; accepted July 31, 1997.
A
ccurate diagnosis of pleural lesions is vital for clinical and medicolegal reasons. Early and precise diagnosis of cytologic material
obtained from effusion fluid or small closed pleural and peritoneal
biopsy samples may influence clinical management and avoid more
invasive diagnostic procedures. Furthermore, from a legal viewpoint,
compensation claims from workers exposed to asbestos demands an
accurate diagnosis of malignant mesothelioma.
The difficulty in distinguishing malignant mesothelioma from a
benign mesothelial proliferation is well recognized, especially when
dealing with small tissue samples obtained by closed needle biopsy.
q 1998 American Cancer Society
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CANCER February 1, 1998 / Volume 82 / Number 3
Diagnosis is a twofold challenge that first involves the
distinction of malignancy from a benign reactive process, or, if malignant, establishment of the tumor type
(usually metastatic adenocarcinoma vs. mesothelioma). This study addresses the first of these problems.
A variety of ancillary tests have been used to assess
mesothelial tissue samples as a means to establish a
diagnosis. These include histochemistry, immunohistochemistry with a wide range of antibodies, and electron microscopy, but as yet no single test has been
found to be reliable to distinguish between malignant
and benign mesothelial tissue.
Epithelial membrane antigen (EMA) is an antibody that has been found to be of value in the distinction between mesothelioma, in which cells usually are
labeled positively with a strong membrane reaction,
and reactive mesothelioses, in which cells usually are
negative, weakly, or diffusely decorated.1 – 3
More recently, measurement of silver-stained nucleolar organizer regions (AgNORs) has been used in
an attempt to demonstrate a distinction between benign and malignant cells. It has been shown that quantitation and area analysis of AgNORs within cells may
discriminate between benign and malignant tissue.
The objective of this investigation was to ascertain
further the value of EMA staining and AgNOR assessment to help with the distinction between benign and
malignant mesothelial lesions, and to evaluate the sensitivity and specificity of the two tests in isolation and
when the results of both were combined.
MATERIALS AND METHODS
Case Retrieval and Cohort Determination
The cases included in this study were retrieved from
paraffin blocks of archival biopsy specimens taken
during the past 20 years and collected from the following Western Australian public hospitals: Sir Charles
Gairdner Hospital, Royal Perth Hospital, Fremantle
Hospital, and from Flinders Medical Centre, South
Australia. Cases were identified by a computer search
of Systematized Nomenclature Of Pathology (SNOP)coded reports of ‘‘malignant mesothelioma’’ of the
pleura and peritoneum; ‘‘suspicious of mesothelioma’’; and benign listings of mesothelial ‘‘proliferation,’’ ‘‘atypia,’’ and ‘‘reaction.’’ SNOP listings yielded
667 benign cases and 430 malignant mesothelioma
cases.
These 1097 cases then were reviewed and rejected
or accepted on the basis of their ability to meet specific
selection criteria that included availability of suitable
tissue, subsequent pathology, and/or clinical followup. Four of the cases reviewed originally were reported
as mesothelioma, but were reclassified as metastatic
carcinoma on the basis of additional immunocyto-
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chemical or ultrastructural evidence on original or
subsequent biopsies or postmortem material. These
cases were excluded from the final test cohort.
No cell block material derived from either fineneedle aspiration samples of solid masses or of effusion samples were included in this study. The tissue
samples used in this study were mainly of the closed
needle biopsy type (Abram’s needle). A few open thoracotomy samples also were included.
Selection Criteria
The histologic diagnosis of malignant mesothelioma
or benign mesothelial hyperplasia was based on previously published, well defined criteria.4 – 6 In summary, malignant mesothelioma was an epithelial
growth, often with a tubulopapillary pattern that demonstrated invasion below the basement membrane
into the stroma. Cytologically the cells may appear
regular or pleomorphic but in all cases morphologic
evidence of invasion with the typical ‘‘raining down’’
of a multipronged growth appearance was determined. Mesothelial hyperplasia was determined to be
a surface proliferation of cuboidal epithelial cells with
no sign of stromal invasion. This was associated often
but not always with a subserosal inflammatory response. Careful consideration was given to the recognition of pseudoinvasion occurring as a result of artefactual displacement, cross-sectioning, and infolding.
Usually the degree of cell proliferation is far less in
hyperplasia than in mesothelioma.
Cases diagnosed as mesothelioma or benign mesothelioses all were followed for at least 3 years. Some
cases had a follow-up of up to 19 years. Acceptance as
a mesothelioma was made on the basis of the original
histology plus further biopsy tissue and/or postmortem diagnosis, acceptance by the Western Australian
Mesothelioma Registry, or by the Australian Mesothelioma Surveillance Programme.7
To optimize the AgNOR test system it is necessary
to have a high level of confidence in the integrity of
the cell-tissue being evaluated. In this case it was determined that there should be at least some epithelial
continuum to ensure that only mesothelial cells were
being tested. On this basis, cases classified as mesothelioma that lacked this continuum (sarcomatous, anaplastic, or simply loose collections of malignant mesothelioma cells within or without fibrous stroma) were
excluded.
This selection process resulted in a cohort of 141
malignant (invasive) mesotheliomas and 73 benign
mesothelial proliferations that were evaluated for
study. The age range of patients with malignant mesothelioma was 27 – 97 years (mean, 59.1 years) and was
16 – 102 years (mean, 52.6 years) for patients with be-
W: Cancer
EMA and AgNOR in Mesothelioma Diagnosis/Wolanski et al.
nign mesothelioses. The male to female ratio was approximately 7:1 for malignant mesothelioma patients
and 2:1 for patients with benign mesothelioses.
Epithelial Membrane Antigen
Immunohistochemistry
Formalin fixed, paraffin embedded 5-mm sections
were investigated for the presence of EMA using the
avidin-biotin complex method.8. After blocking for endogenous peroxidase and clearing for nonspecific
binding by treatment with 3% hydrogen peroxide and
20% normal horse serum, respectively, the primary
antibody, 1:100 EMA (Dakopatts, Copenhagen, Denmark), was applied and incubated at 4 7C overnight.
The bridging antiserum used was biotinylated antimouse immunoglobulin antibody (1:200; Silenus, Melbourne, Australia), which was conjugated with horseradish peroxidase-conjugated streptavidin (1:200;
Silenus). Color was developed by applying diaminobenzidine (Sigma Chemicals, St. Louis, MO), toned by
incubation in CuSO4 , and counterstained with hematoxylin. Slides were dehydrated, cleared, and mounted
in DePeX mounting media (B.D.H. Laboratory Supplies, Poole, Dorset, UK). A case of carcinoma with
proven EMA positivity was used as a positive control,
and a duplicate slide in which primary antisera was
substituted for normal horse serum was used as a negative control.
Scoring and assessment
After staining, all 214 cases were randomized for blind
evaluation. Each case was viewed independently by
two observers who assigned a diagnostic index according to immunoreactivity, stain location, intensity,
and distribution. A stringent scoring process was applied. A positive score was assigned only if definite
brown reaction product was located on the cell membrane.9 A negative score was assigned for faint, nonspecific cytoplasmic staining or when õ 5% of cells
were weakly and specifically stained. An equivocal
score was given if mesothelial cells showed some cytoplasmic or diffuse nonspecific reaction. Although in
some cases of mesothelioma there were areas of diffuse EMA staining, this usually was limited to the more
solid areas of growth and in this study evidence of
clear cut membrane staining was required within the
tissue sample for it to be termed positive. Minimal or
pure diffuse nonmembrane staining was classified as
equivocal and grouped with the negative specimens.
Silver-Stained Nucleolar Organizer Regions
Staining
A modified 1-step silver staining procedure10 was applied to 5-mm sections. All cases included for immuno-
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585
histochemistry also were stained for AgNOR. Slides
were incubated at room temperature in a silver solution prepared by combining at a ratio of 2:1; 2% gelatin
dissolved in a 0.5% solution of 90% formic acid and a
filtered 50% solution of silver nitrate. After incubation
for 60 minutes the silver solution was washed off with
3 rinses of distilled water, the slides fixed in 5% sodium
thiosulfate solution for 5 minutes, and washed 3 times
with distilled water. Sections then were dehydrated,
cleared, and mounted in DePeX mounting medium.
To be included in the AgNOR count and area analysis, staining needed to be clean with minimal precipitation and mesothelial cells had to be identified easily,
with a minimum of 30 cells to be analyzed. Analysis
was impossible on approximately 50% of cases due to
specimen age, fixation, and embedding components
causing unsatisfactory staining. Eighty malignant mesothelioma cases and 26 benign mesothelial proliferations were suitable for analysis.
AgNOR count/area analysis
Slides were randomized and analyzed by a single observer as a blind test. Slides were screened under light
microscopy at 1100 oil immersion and viewed on a
computer monitor via a video camera linked to the
microscope. For each imaged cell, the number of
nucleoli within the nucleus and the number of AgNOR
within each nucleolus also were recorded. Nucleoli
and AgNOR area were measured using a digital image
analysis system (Optimas 4.1; Bioscan Incorporated).
At least 30 cells were selected and counted from each
case. If much variation was observed throughout the
sample, a further 20 cells were counted and included
in calculations. As reported by Orrell et al,11 30 cells
usually were significant to achieve a stable mean. All
data were recorded on a computer data base (Microsoft Excel 5.0; Microsoft Corp., Redmond, WA).
RESULTS
All positive and negative controls used showed acceptable staining patterns. EMA positive reaction was
found in 103 of the 141 malignant mesothelioma cases
studied. Thirty-seven of the 141 malignant cases
showed no immunoreactivity to EMA, and 1 case demonstrated cytoplasmic staining and was classified as
equivocal. No benign cases demonstrated positive
membranous staining, but three cases showed diffuse
cytoplasmic staining and were classified as equivocal.
The remaining 70 cases showed no evidence of immunoreactivity and were classified as negative (Table 1)
(Fig. 1).
AgNOR Results
AgNORs were identified as black, usually spheric particles observed within the nucleolus and also within
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CANCER February 1, 1998 / Volume 82 / Number 3
TABLE 1
EMA Immunoreactivity of Benign and Malignant Mesothelioses
Malignant mesothelioma
Benign mesothelioses
Total
Positive
141
73
103
0
Negative
73.1%
37
70
Equivocal
26.2%
95.9%
1
3
0.7%
4.1%
EMA: epithelial membrane antigen.
TABLE 2
Average AgNOR Count per Cell in Benign and Malignant Mesothelioses
Malignant mesothelioma
Benign mesothelioses
No. of cases
counted
Average no.
AgNOR/cell
SD no.
AgNOR/cell
Range
80
26
6.12
5.03
1.2556
0.8324
4.00–9.54
3.08–6.52
AgNOR: silver-stained nucleolar organizer regions; SD: standard deviation.
FIGURE 1. Immunoreactivity of epithelial membrane antigen (EMA) in
Combined EMA and AgNOR Results
Eighty cases with both EMA and AgNOR analysis were
available for assessment. Combining the 2 results improved diagnostic sensitivity from 78.8% (EMA) and
63.8% (AgNOR), respectively, to 95.0% (combined).
Seventy-six of the 80 proven malignant mesothelioma
cases that were tested for both markers were given a
positive result in at least 1 of the 2 tests. Of the 17 of
80 malignant mesothelioma cases that were negative
for EMA, all but 4 were classified as positive by the
AgNOR area threshold (Fig. 3).
benign and malignant mesothelioses.
DISCUSSION
the nucleus. Occasional dark, nonspecific background
particles were observed but easily were distinguishable
from the AgNORs and were disregarded.
The average number of AgNORs per cell was
highly variable in both benign and malignant groups.
Generally, malignant mesothelioma had a greater
number of AgNORs per cell, but the ranges of AgNOR
counts indicate the large overlap between individual
benign and malignant values (Table 2).
Study of the area occupied by AgNORs per cell
was more discerning. Above a threshold of 0.6677 mm2
AgNOR area per cell all cases were classified as malignant. Fifty-one of 80 malignant cases (63.8%) and no
benign cases had a higher average AgNOR area than
the threshold. The threshold of this test demonstrated
100% specificity and sensitivity was 63.8% (i.e., 29 of
the 80 mesothelioma cases were õ 0.6677mm2) (Table
3) (Fig. 2).
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Difficulty in the differential diagnosis of the often ambiguous reactive mesothelioses from well differentiated epithelial malignant mesothelioma in small biopsy samples is well recognized. Well differentiated
epithelial type malignant mesothelioma cells can appear cytologically bland and show little distinction
from normal cells. Malignancy usually is demonstrated through evidence of extensive cytoproliferation, cytologic atypia, and the presence of invasion.4,5,12 – 14 However, in small sample biopsies and
problem cases, these indications may be absent. As
yet there is no single reliable method or ancillary test
that distinguishes benign from malignant mesothelial
cells in these circumstances.4,13,15 – 17 Although EMA
and AgNOR testing have been used independently, to
our knowledge, a formal combination of the two techniques has not been utilized.
EMA is derived as an antibody to a human milk
fat globule membrane immunogen.18 In the human
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EMA and AgNOR in Mesothelioma Diagnosis/Wolanski et al.
587
TABLE 3
Average AgNOR Area per Cell in Benign and Malignant Mesothelioses
Malignant mesothelioma
Benign mesothelioses
No. of cases
counted
Avg area
AgNOR/cell
SD area
AgNOR/cell
Range
80
26
0.7872
0.5264
0.2178
0.0757
0.3392–1.2620
0.3997–0.6677
AgNOR: silver-stained nucleolar organizer regions; Avg: average; SD: standard deviation.
FIGURE 2.
Average silver-stained nucleolar organizer region (AgNOR)
area per cell in benign and malignant mesothelioses.
FIGURE 3. Combined sensitivity of epithelial membrane antigen (EMA)
and silver-stained nucleolar organizer region (AgNOR) results in invasive
mesothelioma.
lactating breast, lipid is secreted by mammary epithelial cells and is bound by a milk fat globule membrane
that is acquired from the cell on passage from cytoplasm to lumen.19 Antibodies raised against milk fat
globule membrane originally were believed to be
uniquely reactive to breast epithelium but subsequently were found to undergo specific reaction with
simple and glandular epithelium including the respiratory tract, pancreas, intestine, and endometrium.18,20
The EMA reaction product, reflecting cell activation,
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has been demonstrated on the surface of mesothelial
cells at the light microscopic level and ultrastructurally, and appears to quantitatively reflect the amount
of the antigen present on cells.12 Previous studies have
shown that EMA can be detected in small amounts on
benign and reactive mesothelial cells in contrast to the
extensive staining in epithelial-type malignant mesotheliomas. Therefore, EMA has been found to be useful
in suggesting malignancy in cases of mesothelial proliferation, although the specificity and sensitivity of
EMA has differed between studies.1,3,16,21 – 24 Henderson
et al4 and others3,25 reported strong positive results in
a majority of malignant mesothelioma specimens and
only occasional immunoreactivity in benign cases.
In our study, EMA staining showed 73% sensitivity
for malignant mesothelioma (103 of 141), and a specificity of approximately 96%. Three equivocal results
and no false-positive results were identified in 73
proven benign cases. Positive staining was observed
to be restricted mostly to the membrane with stain
intensity influenced by cell growth pattern. Peripheral
cells tended to stain more intensely26; for example,
tubulopapillary areas showed quite extensive membranous staining, whereas little membranous staining
was detected within solid areas. The significance of
nonspecific EMA reactivity is not understood, with antigen cross-reactivity being a possible explanation.9
The one malignant and three benign equivocal cases
showed nonspecific cytoplasmic staining to EMA and
no other distinguishing features. For the purpose of
clarifying the diagnostic suitability of the EMA test,
these cases were grouped with other negative cases.
AgNORs are loops of DNA encoding for ribosomal
RNA, and are important in protein synthesis and the
formation of the nucleolus. These regions are located
on the short arms of acrocentric chromosomes.11,27 – 34
Associated with these organizer regions are argyrophilic, acidic nonhistone proteins that, due to their silver
affinity, can be stained to allow the associated DNA
organizer regions to be visualized.11,28,29,31,35 The significance of AgNORs within a cell has still not been
determined fully, but they generally are accepted as a
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CANCER February 1, 1998 / Volume 82 / Number 3
reflection of proliferation, cell ploidy, or an increase
in transcription.15,32,33,36,37
Studies have shown some correlation between the
average number of AgNORs per cell and malignancy.10,11,28,33,38 – 41 However, a degree of overlap between benign and malignant cases has led some investigators to question the diagnostic value of this
method.28,38 – 41
Previous studies on mesothelial tissue have shown
that the average AgNOR count in malignant mesothelioma is significantly higher than in benign mesothelial
hyperplasia,36,42,43 but overlap between the benign and
malignant reference ranges has cast some doubt on
the use of AgNOR count as a diagnostic discriminant
in single cases.15,43 – 45 In the current study, we also
found the average AgNOR count per cell was higher
in malignant mesothelioma cells than in benign mesothelioses. However, from a diagnostic viewpoint, the
large overlap between benign and malignant AgNOR
counts per cell (4.00 – 9.54 for malignant cells and
3.08 – 6.52 for benign cells) indicates that merely
counting the number of AgNORs per cell is unlikely
to provide a reliable diagnostic discriminant.
More recently, it has been suggested that the area
occupied by AgNORs may be a more useful diagnostic
discriminant.33,46,47 In a study on mesothelial cells, Lim
et al36 reported that malignant mesothelioma samples
from peritoneal effusions had a higher mean AgNOR
area per cell than benign specimens and the researchers were able to assign a cutoff area value of 1.1 mm2
that enabled a diagnosis of malignancy. Derenzini et
al48 also found average AgNOR area per cell was higher
in malignant than benign mesothelioses in pleural
fluids.
Our results show that AgNOR area was of greater
diagnostic significance than AgNOR count. Above a
threshold of 0.6677 mm2 area per cell, all cases were
malignant. All benign cases had a lower AgNOR area
per cell than the threshold (100% specificity), and 64%
of malignant cases had average AgNOR areas above
the threshold. We concluded that any case with a measured average AgNOR area per cell greater than this
threshold would indicate malignancy, regardless of
immunohistochemical and morphologic findings.
The use of the assessment of AgNORs in diagnosis
has not been accepted universally. Varying methods
of AgNOR staining, especially the method of fixation
and staining time have been shown to affect AgNOR
area and count49,50 and demonstrate that it is essential
for a standard technique to be accepted for comparative studies. The test also may be limited by techniques
used for the fixation and embedding of samples that
may prevent adequate staining and analysis. We believe that standard methods and the use of computer-
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linked image analysis may improve the accuracy and
reproducibility by increasing the objectivity of measurements.
Neither EMA immunoreactivity nor AgNOR area
analysis alone enables distinction of a single case of
benign mesothelioses from malignant mesothelioma.
Combining these techniques enabled 96% of cases to
be assessed accurately. Four cases of 80 gave a falsenegative diagnosis and no cases falsely indicated malignancy. The four malignant cases that showed both
nonreactivity to EMA and an AgNOR area lower than
the malignant threshold demonstrated no other distinguishing histologic features.
This study suggests that the use of EMA and AgNOR
as conjoint ancillary tests in the differential diagnosis
of difficult pleural biopsies may have a role in distinguishing benign reactions from malignant proliferations.
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