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© 2017 College of American Pathologists

 Review Article
Application of Immunohistochemistry in the Diagnosis
of Pulmonary and Pleural Neoplasms
Jennifer S. Woo, MD; Opal L. Reddy, MD; Matthew Koo, MD; Yan Xiong, MD; Faqian Li, MD, PhD; Haodong Xu, MD, PhD
 Context.—A vast majority of neoplasms arising from lung
or pleura are initially diagnosed based on the histologic
evaluation of small transbronchial, endobronchial, or
needle core biopsies. Although most diagnoses can be
determined by morphology alone, immunohistochemistry
can be a valuable diagnostic tool in the workup of
problematic cases.
Objective.—To provide a practical approach in the
interpretation and immunohistochemical selection of lung/
B iopsies of lung and pleural neoplasms play an important
role in the characterization and staging of lung cancers,
particularly for patients with advanced disease (stages III
and IV) and for patients who are not candidates for surgical
procedures. Clinical and imaging correlation in conjunction
with morphologic assessment by routine hematoxylin-eosin
staining is crucial in the interpretation of small biopsies.
However, immunohistochemistry (IHC) may be a valuable
diagnostic tool in the workup of challenging cases. For
example, it can help differentiate between lung adenocar-
cinoma and squamous cell carcinoma (SqCC), lung adeno-
carcinoma and malignant mesothelioma (MM), primary and
metastatic carcinomas, and small cell lung carcinoma
(SCLC) and carcinoid tumor. It can also help detect
oncogenic mutants, such as endothelial growth factor
receptor (EGFR), anaplastic lymphoma kinase (ALK), and
repressor of silencing 1 (ROS1), and immune checkpoint
molecules, such as programmed death ligand-1 (PD-L1).
This article provides an up-to-date review of the role of IHC
in the workup of common entities seen in the small lung/
pleural biopsy setting while using examples.
Accepted for publication December 15, 2016.
From the Department of Pathology and Laboratory Medicine,
David Geffen School of Medicine at UCLA, Los Angeles, California
(Drs Woo, Reddy, Koo, and Xu); the Department of Pathology, Peking
University First Hospital, Beijing, China (Dr Xiong); and the
Department of Laboratory Medicine and Pathology, University of
Minnesota, Minneapolis (Dr. Li).
The authors have no relevant financial interest in the products or
companies described in this article.
Presented at the First Chinese American Pathologists Association
(CAPA) Diagnostic Pathology Course: Best Practices in Immunohis-
tochemistry in Surgical Pathology and Cytopathology; Flushing, New
York; August 22–24, 2015.
Reprints: Haodong Xu, MD, PhD, Department of Pathology and
Laboratory Medicine, David Geffen School of Medicine at UCLA,
10833 Le Conte Ave, CHS 13-145E, Los Angeles, CA 90095-1732
(email: HaodongXu@mednet.ucla.edu).
Arch Pathol Lab Med
pleura–based neoplasms obtained from small biopsy
samples.
Data Sources.—A literature review of previously pub-
lished articles and the personal experience of the authors
were used in this review article.
Conclusion.—Immunohistochemistry is a useful diag-
nostic tool in the workup of small biopsies from the lung
and pleura sampled by small biopsy techniques.
(Arch Pathol Lab Med. doi: 10.5858/arpa.2016-0550-RA)
PRIMARY EPITHELIAL TUMORS OF LUNG
An 82-year-old nonsmoking man presented with a 4.7 3
3.6-cm right upper lobe mass by computed tomography
(CT) chest imaging. A CT-guided biopsy demonstrated a
sheetlike proliferation of poorly differentiated malignant
epithelial cells (Figure 1, A). The tumor cells were positive
for p40 (Figure 1, B) and cytokeratin 5/6 (CK5/6) (Figure 1,
C), but negative for thyroid transcription factor 1 (TTF-1)
(Figure 1, D) and napsin A (Figure 1, E). The final diagnosis
was poorly differentiated SqCC.
A 66-year-old Asian woman presented with a poorly
circumscribed, solid 2.6 3 2.5-cm right upper lobe mass by
CT chest imaging. A CT-guided biopsy demonstrated a solid
growth pattern of poorly differentiated malignant epithelial
cells (Figure 2, A). The tumor cells were positive for TTF-1
(Figure 2, B) and napsin A (Figure 2, C), but negative for p40
(Figure 2, D) and CK5/6 (Figure 2, E). The final diagnosis
was poorly differentiated adenocarcinoma with a solid
pattern of growth. Molecular studies showed that the tumor
cells were positive for an EGFR exon 19 deletion.
Historically, the most clinically significant distinction
among lung tumors was the distinction between non–small
cell lung carcinoma (NSCLC) and SCLC. However, because
of markedly different prognostic and treatment implications
among different lung tumors, accurate subtyping with
molecular characterization is critical. This is especially true
for the most common NSCLC types, lung adenocarcinoma
and SqCC, as treatment options and the role of ancillary
molecular and cytogenetic studies widely differ for both
tumors. Specifically, EGFR inhibitors such as erlotinib (OSI
Pharmaceuticals, Melville, New York; Hoffmann-La Roche,
Basel, Switzerland; Genentech, South San Francisco, Cal-
ifornia) and gefitinib (AstraZeneca, London, United King-
dom) have been shown to be effective in EGFR-mutated
tumors,1 and the ALK inhibitor crizotinib (Pfizer, New York,
New York) has been shown to be effective in tumors with
EML4-ALK fusion,2 which are both predominantly seen
with adenocarcinomas. Additionally, several therapeutic
Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al 1
Figure 1. Poorly differentiated squamous cell carcinoma of the lung. The needle core biopsy shows sheets of malignant large cell cells without
obvious squamous differentiation (A). The tumor cells are positive for p40 (B) and cytokeratin 5/6 (C). The tumor cells are negative for both thyroid
transcription factor 1 (D) and napsin A (E) (hematoxylin-eosin, original magnification 3400 [A]; original magnification 3400 [B through E]).

2 Arch Pathol Lab Med Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al

Figure 2. Poorly differentiated adenocarcinoma of the lung. The needle core biopsy shows a solid pattern of malignant large cells without glandular
formation (A). The tumor cells are positive for thyroid transcription factor 1 (B) and napsin A (C). The tumor cells are negative for both p40 (D) and
cytokeratin 5/6 (E) (hematoxylin-eosin, original magnification 3400 [A]; original magnification 3400 [B through E]).

Arch Pathol Lab Med Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al 3

 Table 1. Common Immunohistochemical Stains
Used to Differentiate Pulmonary Squamous Cell
Carcinoma From Adenocarcinoma
Squamous Cell
Carcinoma Adenocarcinoma
CK5/6 TTF-1–SPT24 (lower specificity)
p40 (higher specificity) TTF-1–8G7G3/1 (higher specificity)
p63 (lower specificity) Napsin A
CK903/34bE12
Abbreviations: CK, cytokeratin; TTF-1, thyroid transcription factor 1.
agents used in the treatment of adenocarcinoma are
contraindicated in SqCC, including pemetrexed (Alimta,
Eli Lilly and Company, Indianapolis, Indiana), because of
the lack of effectiveness,3 and the vascular endothelial
growth factor (VEGF) inhibitor bevacizumab (Avastin,
Genentech), because of the risk of life-threatening pulmo-
nary hemorrhage.4 Two genetic alterations have been
recently identified in pulmonary SqCC, discoidin domain
receptor tyrosine kinase 2 (DDR2) mutation5 and fibroblast
growth factor receptor 1 (FGFR1) amplification,6 both of
which have potential as novel therapeutic targets.
In most instances, the morphologic distinction between
adenocarcinoma and SqCC is relatively straightforward: the
formation of glands and the production of cytoplasmic
mucin are characteristic of adenocarcinoma, whereas the
production of keratin and the presence of intercellular
bridges are characteristic of SqCC. However, the pathologist
may run into diagnostic challenges in the setting of a poorly
differentiated tumor, or tumors with nonspecific morpho-
logic findings, especially in the biopsy setting. In this case,
the judicious use of IHC markers may be helpful for further
characterization (Table 1).
The most useful IHC markers for pulmonary adenocar-
cinoma include TTF-1 and napsin A. TTF-1 is a homeodo-
main-containing transcription factor that is predominantly
found in normal type II alveolar pneumocytes.7 With a
reported sensitivity ranging from 75% to 80%,8,9 TTF-1 has
long been the predominant nuclear IHC marker used to
identify cells of lung origin. However, TTF-1 expression has
been shown to decrease inversely with the degree of tumor
differentiation.8–10 Of the 2 main commercial TTF-1 mono-
clonal antibodies available for IHC staining (8G7G1/1 and
SPT24), the SPT24 clone has been shown to have a stronger
affinity for TTF-1. However, the SPT24 TTF-1 clone may also
show an affinity for colorectal adenocarcinomas.11
Napsin A, a relatively new cytoplasmic IHC marker for
pulmonary adenocarcinoma, is an aspartic proteinase
involved12 in the maturation of surfactant protein B that
has an expression thought to be regulated by TTF-1.13
Napsin A has been shown to be superior to TTF-1 in
distinguishing primary lung adenocarcinomas from other
carcinomas (except for renal cell carcinoma,14,15 clear cell
carcinomas of the gynecologic tract,16,17 and thyroid
carcinomas15). Napsin A, with a higher specificity but a
lower sensitivity for adenocarcinoma than TTF-1, was also
found to be a useful marker in cases of poorly differentiated
lung adenocarcinoma or an unknown primary tumor.17
Another IHC marker for glandular origin may be the
cytoplasmic immunostain cytokeratin 7 (CK7). However,
CK7 reactivity can be seen in most poorly differentiated
adenocarcinomas and has been reported in 9 of 15
morphologically challenging cases of poorly differentiated
SqCCs (60%), which limits its diagnostic utility.18
4 Arch Pathol Lab Med
Immunohistochemical markers for SqCC include p63,
CK5/6, cytokeratin 34bE12 (CK903), and p40. The p63
antibody, with numerous studies showing excellent sensi-
tivity as a marker for SqCC,19–21 has long been the most
commonly used nuclear marker for squamous origin.
However, p63 has been shown to be positive in 16% to
65% of lung adenocarcinoma20,22–24 and in 55 of 172 cases
(32%) of diffuse large B cell lymphoma.25
p63 consists of several isoforms. The 2 major groups
include DNp63 and TAp63.26 DNp63 is the predominant p63
transcript in SqCC of lung and functions as an oncogene,27,28
whereas TAp63 has been shown to function as a p53-like
tumor suppressor.26 In the majority of pathology laborato-
ries, the routine p63 antibody is 4A4, which recognizes both
TAp63 and DNp63 isoforms. The p40 antibody, which
exclusively recognizes the DNp63 isoform, may have a
superior specificity29 but inferior sensitivity30–32 compared
with p63 in the diagnosis of pulmonary SqCC.
Previous studies have demonstrated that a majority of
poorly differentiated NSCLCs can be subclassified as
adenocarcinoma or SqCC by IHC using a simple panel of
IHC markers.19,33,34 For limited biopsy samples, we recom-
mend an initial IHC panel of TTF-1 and p40. If needed,
additional squamous markers (p63 and CK5/6) or glandular
markers (napsin A and CK7) may be added. The distinction
between adenocarcinoma and SqCC is generally straightfor-
ward when following this IHC approach. One or more
markers of glandular differentiation (TTF-1 or napsin A) with
negative squamous markers is supportive of adenocarcinoma.
Conversely, one or more positive squamous markers (p40 or
p63), in the context of negative glandular markers, is
suggestive of SqCC. Cases of TTF-1– and/or napsin A–
positive adenocarcinoma may show focal p63 expression.19
Interestingly, diffuse coexpression of TTF-1 and p63 may be
seen in adenocarcinomas with signet ring cell morphology
(many of which contain EML4-ALK translocations).35 Ad-
enosquamous carcinoma requires the presence of 2 separate
components demonstrating opposite and mirror-image
staining patterns (napsin A and TTF-1 positive and p40 and
CK5/6 negative in the glandular component, with napsin A
and TTF-1 negative and p40 and CK5/6 positive in the
squamous component).36 Nevertheless, in small biopsy
samples, the ability to definitively diagnose adenosquamous
carcinoma may be particularly challenging.
Isolated p63 positivity in scattered cells is a nonspecific
staining pattern that does not contribute to the further
classification of the lesion. Similarly, isolated CK5/6 staining
in scattered cells, in the absence of positivity for additional
squamous or glandular markers, is also a nonspecific
finding. In these cases, or in cases with nonreactivity to
the entire proposed IHC panel, the most suitable diagnosis
would be NSCLC, not otherwise specified.37
A major pitfall in interpreting IHC-stained slides of small
lung biopsy samples is the positivity of normal alveolar
epithelium for TTF-1 and/or napsin A. Therefore, the proper
interpretation of immunomarkers requires morphologic
correlation. In addition, alveolar macrophages show cyto-
plasmic napsin A staining. Prior to the availability of IHC
stains, pathologists heavily relied on the histochemical
mucin stain to confirm a diagnosis of adenocarcinoma. Even
today, despite a low sensitivity,38–40 special stains for
cytoplasmic mucin may still prove to be useful in particularly
challenging cases that show nonspecific staining patterns.
Furthermore, in this age of targeted therapies, molecular
and cytogenetic studies play a critical role in the workup of
Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al
primary lung malignancies, especially in NSCLC. Although
molecular and cytogenetic testing will remain the gold-
standard methodology for the detection of specific somatic
genetic mutations, IHC has emerged as a fairly effective and
rapid means for identifying these genetic variants. One of
the most significant mutations in NSCLC includes sensitiz-
ing mutations of EGFR, particularly in exons 18–21. EGFR
mutations are important to identify, as neoplasms demon-
strating these mutations may be susceptible to EGFR–
tyrosine kinase inhibitor (TKI) therapy (ie, erlotinib;
gefitinib; afatinib, Boehringer Ingelheim, Ingelheim, Ger-
many]). The 2 most common EGFR mutations include in-
frame deletions in exon 19 (E746_A750del) and a point
mutation at codon 858 in exon 21 (L858R), together
representing 85% to 90% of EGFR mutations in NSCLC
patients.41,42 Mutation-specific antibodies for these 2 genetic
mutations were developed43 in 2009, and numerous studies
have since demonstrated that IHC may be used as a reliable
prescreening test for detecting EGFR mutations in
NSCLC.44–47 A moderately differentiated adenocarcinoma
of the lung shows an acinar pattern of growth (Figure 3, A)
and contains an EGFR mutant with E746-A750 (Figure 3, B)
detected by using rabbit monoclonal antibody against EGFR
E746-A750 (clone 6B6; Cell Signaling Technology, Danvers,
Massachusetts); a poorly differentiated adenocarcinoma of
the lung exhibits a solid pattern of growth (Figure 4, A) and
harbors an EGFR mutant with L835R (Figure 4, B) detected
by using rabbit monoclonal antibodies against EGFR L835R
(clone 43B2; Cell Signaling Technology).
Chromosomal rearrangements of the ALK gene, although
found in a minority (0.4%–15%) of NSCLCs,48,49 are also
important to identify, as these tumors may be susceptible to
ALK inhibitors such as crizotinib. Recent studies have also
shown that the use of ALK antibodies may also be an
effective prescreening tool to detect the presence of ALK
rearrangements in addition to the conventional ALK
fluorescence in situ hybridization testing.50,51 A moderately
differentiated adenocarcinoma of the lung shows an acinar
pattern of growth (Figure 5, A) and expresses ALK protein
(Figure 5, B) detected by using a rabbit monoclonal antibody
against ALK (clone D5F3; Cell Signaling Technology).
Immunohistochemical stains have also been developed to
detect rearrangements of ROS1 (present in 1%–2% of
NSCLC),52,53 which encodes for a receptor tyrosine kinase.
Several studies suggest that ROS1 may represent another
therapeutic target of the ALK inhibitor crizotinib,52–54 with a
recent study showing a marked antitumor activity by
crizotinib in patients with ROS1-rearranged advanced
NSCLC.55 Similar to IHC for ALK, IHC stains for ROS1
have been shown to be an effective and cost-effective means
to screen for ROS1 rearrangements.56–58 A poorly differen-
tiated adenocarcinoma of the lung shows acinar and
micropapillary patterns of growth (Figure 6, A) and
expresses ROS1 (Figure 6, B), detected by using a rabbit
monoclonal antibody against ROS1 (clone D4D6; Cell
Signaling Technology). Therefore, the judicious use of
mutation-specific IHC stains may be used as a practical
screening tool to detect certain actionable mutations (EGFR,
ALK, and ROS1) amenable to targeted therapy.
In addition to the ROS1, ALK, and EGFR pathways,
programmed death receptor-1 (PD-1), an immunoregula-
tory receptor expressed by activated T cells,59 and its ligand,
PD-L1 in cancer cells, have been shown to be promising
targets in NSCLC. Targeted therapies against PD-1 have
been postulated to treat cancers by restoring proper effector
Arch Pathol Lab Med
T-cell function. Recent clinical trials have shown that anti–
PD-1 therapy in NSCLC demonstrates antitumor activity in
patients with advanced NSCLC.60–64 The US Food and Drug
Administration has approved inhibitors targeting PD-1 for
patients with NSCLC. Currently, a number of different anti–
PD-L1 antibodies by various vendors have been formally
studied. In the future, pathologists may expect to become
more familiar with PD-L1 as an important NSCLC
predictive or companion biomarker. Figure 7, A, shows a
pleomorphic carcinoma with a prominent spindle cell
component that is strongly positive for TTF-1 (Figure 7, B)
and PD-L1 (Figure 7, C) by using a mouse monoclonal
antibody against PD-L1 (clone 22C3; DAKO, Carpinteria,
California).
PLEURAL MM VERSUS LUNG CARCINOMA
A 60-year-old man presented with multiple right pleural
masses on CT imaging. A right pleural biopsy was
performed at an outside institution and a diagnosis of
epithelioid MM was rendered. The patient was evaluated for
a decortication of pleural mesothelioma and a rebiopsy was
performed. Biopsies of the right pleural masses demon-
strated a biphasic MM (75% epithelioid type, 25% sarco-
matoid type) (Figure 8, A). The tumor cells were positive for
AE1/AE3 (Figure 8, B), calretinin (Figure 8, C), and Wilms
tumor 1 (WT-1) (Figure 8, D).
Malignant mesothelioma of the pleura is a rare neoplasm
arising from mesothelial cells. Although known for a great
diversity of histologic patterns, MMs are generally divided
into 3 major histologic types: epithelioid, sarcomatoid, and
biphasic (mixed epithelioid and sarcomatoid).65 Given the
wide range of morphologic features, the differential
diagnosis of MM may significantly vary depending on the
histologic type. For epithelioid MM, the differential diag-
nosis may include carcinoma (including primary adenocar-
cinoma, metastatic adenocarcinoma, or SqCC) as well as
other tumors with epithelioid features. Sarcomatoid MM
raises the differential diagnosis of sarcomatoid carcinoma,
malignant tumors with sarcomatoid features, and a variety
of malignant sarcomas, including undifferentiated pleomor-
phic sarcoma (formerly malignant fibrous histiocytoma),
osteosarcoma, and chondrosarcoma. Lymphoma, melano-
ma, angiosarcoma, and epithelioid hemangioendothelioma
are also on the differential diagnosis, as are benign
mesothelial proliferations. Biphasic patterns should raise
the differential diagnosis of other biphasic tumors such as
synovial sarcoma.
The most important consideration for the pathologist is
the patient’s clinical and radiologic features in correlation
with the hematoxylin-eosin morphology. However, IHC can
be helpful in confirming MM, and the initial selection of
stains will vary depending on the histologic type. In general,
nearly all mesothelial cells (including mesotheliomas) are
positive for pancytokeratin (including AE1/AE3).66 However,
sarcomatoid MM may show loss of keratin reactivity. In
these cases, a cocktail of keratins may improve detection.67
Furthermore, sarcomatoid MM with either osteosarcoma-
tous or chondrosarcomatous differentiation is known to be
typically negative for keratin staining.
Other mesothelial markers include calretinin (strong and
diffuse nuclear and cytoplasmic staining), CK5/6 (cytoplas-
mic staining), D2-40 (membranous staining), and WT-1
(nuclear staining).66 It must be noted that because of the
variability of staining qualities among different antibody
Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al 5
Figure 3. Moderately differentiated adenocarcinoma of the lung. The biopsy shows a moderately differentiated adenocarcinoma with an acinar
pattern (hematoxylin-eosin, A). The tumor cells express the EGFR mutant protein, E746-A750 (B) (hematoxylin-eosin, original magnification 3200
[A]; original magnification 3200 [B]).
Figure 4. Poorly differentiated adenocarcinoma of the lung. The biopsy shows poorly differentiated adenocarcinoma with a solid pattern (A). The
tumor cells express the EGFR mutant protein L858R (B) (hematoxylin-eosin, original magnification 3200 [A]; original magnification 3200 [B]).

clones and IHC laboratories, no single specific panel of IHC
antibodies can be recommended universally. In general, the
pathologist should become familiar with the staining pattern
at his or her performing laboratory and take into account
this knowledge when selecting the initial panel of stains.
Immunohistochemistry laboratories should aim for a
sensitivity of at least 80% when validating immunostains
for this purpose.68
6 Arch Pathol Lab Med
For the initial workup of an epithelioid MM, where the
main differential diagnoses are MM versus carcinoma, a
reasonable panel may include 2 mesothelial markers and 2
carcinoma markers (Table 2). An expanded panel may be
necessary if the initial panel reveals discordant results or if a
metastatic adenocarcinoma is suspected. Immunostains
positive for adenocarcinoma include TTF-1, napsin A, Ber-
EP4, carcinoembryonic antigen (CEA), Leu-M1, and MOC-
Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al
Figure 5. Moderately differentiated adenocarcinoma of the lung. The biopsy shows a moderately differentiated adenocarcinoma with an acinar
pattern (A). The tumor cells express ALK protein (B) (hematoxylin-eosin, original magnification 3200 [A]; original magnification 3200 [B]).
Figure 6. Poorly differentiated adenocarcinoma of the lung. The biopsy shows a poorly differentiated adenocarcinoma with acinar and
micropapillary patterns (A). The tumor cells express ROS1 protein (B) (hematoxylin-eosin, original magnification 3200 [A]; original magnification
3200 [B]).

31. TTF-1 and napsin A have the additional benefit of
helping to confirm a primary lung origin. In differentiating
MM from adenocarcinoma, calretinin, CK5/6, D2-40, and
WT-1 are very useful mesothelial markers. However, in
differentiating MM from SqCC, the role of mesothelial
markers is more limited, owing to the fact that SqCCs may
be positive for CK5/6, D2-40, and (to a lesser extent)
calretinin. Therefore, WT-1 is the most clinically useful
immunostain in differentiating MM from SqCC. Markers
Arch Pathol Lab Med
positive in SqCC include p40, p63, MOC-31, and Ber-EP4.69
Additional immunomarkers that may be helpful, depending
on the differential diagnosis, include CD45 (for lymphoma);
S100, HMB-45, MART1, and SOX10 (for melanoma; both
S100 and SOX10 are more sensitive for spindle cell
melanoma)70; and CD31, CD34 and ERG1 (for angiosarco-
ma and epithelioid hemangioendothelioma).
For sarcomatoid MM, an initial panel may include
multiple cytokeratins, calretinin, and D2-40. Multiple
Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al 7
 keratin antibodies including AE1/AE3, CAM 5.2, and CK7
are recommended, as cytokeratin expression may be absent
or variable in sarcomatoid mesothelioma.71,72 However,
because sarcomatoid carcinoma and metastatic sarcomatoid
renal cell carcinoma may show the same staining pattern,
diffuse keratin positivity with sarcomatoid morphology is
not by itself diagnostic for sarcomatoid mesothelioma.
Metastatic renal cell carcinoma may be highlighted with
paired box gene 2 (PAX2) or PAX8 staining. Moreover, focal
keratin positivity must be interpreted with caution, as a
number of sarcomas may demonstrate focal keratin
positivity.67 For this reason, markers for sarcomatous
differentiation, such as smooth muscle actin (SMA), desmin,
and myoglobin, may be helpful.
Useful markers for sarcomatoid MM include calretinin
and D2-40,73,74 although the number of tumor cells positive
for these markers is variable. Additional immunostains to
distinguish between MM and other sarcomas may include
SMA, desmin, myoglobin and myogenin. Overall, no single
immunostain by itself is particularly helpful for the diagnosis
of sarcomatoid mesothelioma. A carefully chosen panel of
immunostains is the key in the workup of such cases.
Immunohistochemical staining with equivocal or nondiag-
nostic results may benefit from ultrastructural examination
by electron microscopy. However, the diagnostic utility of
electron microscopy may be more limited in the setting of
poorly differentiated tumors.75,76
Benign mesothelial proliferations are also in the differen-
tial diagnosis of MM. Morphologic features favoring
malignancy include dense cellularity, complex papillae,
tubules, cellular stratification, and necrosis.67 However,
because benign and malignant mesothelial cells may share
overlapping cytologic features, the presence of stromal
invasion is considered the most reliable feature for
separating benign versus MM proliferations.77–80 The inva-
sion of stroma or fat by atypical mesothelial cells may be
highlighted by immunostains including AE1/AE3 or calre-
tinin. In this case, the pathologist must determine whether
mesothelial cells represent entrapped benign cells or benign
cells cut en face versus true invasive malignancy. To
distinguish fatlike spaces that may be seen in organizing
pleuritis,81 S100, laminin, and collagen IV stains may be
performed to highlight adipocytes, whereas fatlike spaces
will be negative.
Immunohistochemical stains to distinguish between
benign and malignant mesothelial proliferations may be
helpful, but are often not entirely definitive for malignancy
or benignity (Table 3). Epithelial membrane antigen (EMA)
and p53 have been described as immunomarkers for
malignancy,82–85 whereas desmin has been described as an
indicator of benign mesothelial cells.84,85 A review by King et
al84 demonstrated sensitivity and specificity of desmin and
EMA to be less than 90%, which may be deemed not
sufficient enough when distinguishing benign from malig-
nant.
Several recent studies have reported insulin-like growth
factor II messenger ribonucleic acid–binding protein 3

Figure 7. Pleomorphic carcinoma with prominent spindle cell

component of the lung. The biopsy shows a proliferation of malignant
spindle cells (A), with tumor cells positive for thyroid transcription
factor 1 (B). The tumor cells are strongly positive for programmed death
receptor-1 (C) (hematoxylin-eosin, original magnification 3200 [A];
original magnification 3200 [B and C]).

8 Arch Pathol Lab Med Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al

Figure 8. Biphasic pleural malignant mesothelioma. The biopsy shows a sheet of malignant epithelioid cell proliferation with focal malignant spindle
cells (A). The tumor cells are positive for AE1/AE2 (B), calretinin (C), and Wilms tumor 1 (D) (hematoxylin-eosin, original magnification 3400 [A];
original magnification 3400 [B through D]).

(IMP3) and glucose transporter 1 (GLUT1) to be useful
biomarkers for differentiating MM from reactive mesothelial
cells. IMP3 is an oncofetal protein involved in embryogen-
esis. GLUT-1 is a member of the GLUT family of passive
carriers, which functions as an energy-independent system
for transport of glucose. Both IMP3 and GLUT1 have been
reported in a variety of carcinomas.86,87 Multiple studies
suggest that both epithelioid and sarcomatoid MMs are
more likely to be positive for IMP3 (diffuse dark brown
cytoplasmic staining), whereas reactive mesothelial prolif-
erations are more likely to negative for IMP3.88–91 A
consistent difference in staining intensity of IMP3 between
Arch Pathol Lab Med
benign and malignant proliferations has not been report-
ed.88
Overall, positive staining for IMP3 and/or GLUT1 may be
helpful to confirm the diagnosis of malignancy; however,
negative staining for IMP3 and/or GLUT1 does not
completely rule out MM. BRCA1-associated protein 1
(BAP1) has most recently emerged as a promising biomark-
er for pleural MM, with loss of nuclear BAP1 staining seen
in some mesotheliomas, but none of the benign cases.92,93
BAP1 IHC has been shown to have a relatively high
specificity (100%) but low sensitivity (27%).93
Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al 9
 Table 2. Common Immunohistochemical Stains
Used to Differentiate Pulmonary Malignant
Mesothelioma From Adenocarcinoma
Malignant Mesothelioma Adenocarcinoma
CK5/6 TTF-1
Calretinin Napsin A
D2-40 Ber-EP4
WT-1 B72.3
CEA
Leu-M1
MOC-31
Abbreviations: CEA, carcinoembryonic antigen; CK, cytokeratin; TTF-1,
thyroid transcription factor-1; WT-1, Wilms tumor 1.
Homozygous p16 (CDKN2A) gene deletion identified
using fluorescence in situ hybridization techniques has also
been described as an indicator of malignancies, including
MM.94,95 However, fluorescence in situ hybridization testing
has a number of limitations, such as artifactual loss of p16
due to section thickness of formalin-fixed, paraffin-embed-
ded tissue (tumor cells may not be in the plane of section
and may appear to lose one or more copies of the gene) and
difficulty of picking out individual cells of interest.80
Therefore, a fresh nodule of cells selected for fluorescence
in situ hybridization studies may be more reliable. Addi-
tionally, homozygous p16 deletion is present in only a
subset of mesotheliomas. Therefore, the absence of p16
gene deletion cannot entirely exclude the diagnosis of
malignancy.96
In summary, the role of IHC can be helpful to confirm a
diagnosis of MM, and the precise IHC panel should be
selected with a careful consideration of the differential
diagnosis. However, caution should be made when inter-
preting immunostains, because of staining variability among
different antibody clones and individual IHC laboratories.
Because of this, IHC laboratories should aim for a sensitivity
of at least 80%.67 Electron microscopy and cytogenetic
studies may be helpful in difficult circumstances, although
their diagnostic ability may be limited in the setting of
poorly differentiated tumors.
PRIMARY LUNG TUMORS VERSUS METASTASIS
A 76-year-old woman with a remote history of breast
carcinoma and bilateral lung adenosquamous carcinoma
presented with an enlarging pleural nodule at the lingula. A
CT-guided biopsy of the pleural nodule revealed adenocar-
cinoma (Figure 9, A) positive for GATA-binding protein 3
(GATA3) (Figure 9, B), progesterone receptor (PR) (Figure 9,
C), estrogen receptor (ER) (Figure 9, D), mammaglobin
(Figure 9, E), and gross cystic disease fluid protein 15
(GCDFP-15) (focal), but negative for TTF-1 (Figure 9, F) and
p40. The overall findings were consistent with metastatic
breast carcinoma to the pleura.
Metastases from the extrapulmonary sites represent the
most common form of pulmonary neoplasm, as the lungs
are one of the most common sites of distant metastases.97
Accordingly, although pathologists may more frequently
encounter primary lung neoplasms, differentiating primary
lung malignancies from metastatic neoplasms, especially
from poorly differentiated extrapulmonary adenocarcino-
mas, can be particularly challenging.
TTF-1 and napsin A are 2 widely used markers to confirm
adenocarcinomas of pulmonary origin.15,98 TTF-1 is a
nuclear protein involved in the organogenesis of the lung
10 Arch Pathol Lab Med
Table 3. Common Immunohistochemical Stains
Used to Differentiate Pulmonary Malignant
Mesothelioma From Reactive Mesothelial cells
Malignant Reactive
Mesothelioma Mesothelial Cells
p53 Desmin
EMA IMP3—less frequently
positive
IMP3—frequently positive GLUT1—less frequently
positive
GLUT1—frequently positive
p16 gene homozygous
deletion
BAP1 (loss of nuclear stain)
Abbreviations: BAP1, BRCA1 associated protein 1; EMA, epithelial
membrane antigen; GLUT1, glucose transporter 1; IMP3, IG2BP3,
insulin-like growth factor mRNA-binding protein.
and thyroid, and is expressed in approximately 75% of lung
adenocarcinomas.99 However, it is also highly sensitive and
specific for thyroid carcinomas, with the exception of
anaplastic carcinoma and a small proportion of other
extrapulmonary adenocarcinomas (including ovarian serous
carcinomas, endometrial and endocervical adenocarcino-
mas, and colonic adenocarcinomas).99,100 Napsin A is
predominantly expressed in the lung and kidney, and
demonstrates granular and cytoplasmic staining.101 It has a
comparable sensitivity to that of TTF-1 in identifying lung
adenocarcinomas. It is important to be aware that the 2
markers may not always be coexpressed.101 Similar to TTF-1,
napsin A is not entirely specific for lung adenocarcinomas,
as its expression is seen in a majority of papillary renal cell
carcinomas (75%–80%), a subset of clear cell renal cell
carcinomas (approximately 30%), rare cases of papillary
thyroid carcinomas, and a significant proportion of ovarian
and endometrial clear cell carcinomas.15,100,102 Therefore,
although TTF-1 and napsin A may be used to support a
diagnosis of primary lung adenocarcinoma, in cases where
morphology or clinical history may implicate an extrapul-
monary primary, other organ-specific biomarkers may be
needed.
The above clinical example describes the frequent
challenge of differentiating a primary lung adenocarcinoma
from a metastatic breast carcinoma. Women with breast
cancer have a 30% higher risk than the general population
of developing a second primary malignancy, with approx-
imately 4% to 9% developing lung cancer.103 In this case, a
panel of TTF-1, napsin A, mammaglobin, GCDFP-15, and
GATA3 was used to support the diagnosis of breast
carcinoma metastatic to the lung. Both TTF-1 and napsin
A have been shown to be negative in breast adenocarcino-
ma.103 Mammaglobin and GCDFP-15 are established
cytoplasmic markers for metastatic breast carcinomas, with
positivity in 85% and 53% of breast carcinomas and up to
17% and 2% of lung carcinomas, respectively.9 GATA3, a
nuclear marker, was found to be expressed in 90 of 99 of
breast ductal carcinomas (91%) and 48 of 48 breast lobular
carcinomas (100%), but was also seen in 62 of 72 urothelial
carcinomas (86%),104 61 of 62 basal cell carcinomas of the
skin (98%), 25 of 31 SqCCs of the skin (81%), 11 of 11
choriocarcinomas (100%), 6 of 6 endodermal sinus tumors
(100%), 37 of 64 MMs (58%), 18 of 22 extra-adrenal
paragangliomas (82%), and 22 of 24 pheochromocytomas
(92%).105 GATA3 expression may also have prognostic
implications in patients with breast cancer, where some
Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al
Figure 9. Metastatic breast carcinoma of the lung. The biopsy shows a moderately differentiated adenocarcinoma with glandular formation (A). The
tumor cells are positive for GATA3 (B), progesterone receptor (C), estrogen receptor (D), and mammaglobin (E). The tumor cells are negative for
thyroid transcription factor 1 (F) (hematoxylin-eosin, original magnification 3200 [A]; original magnification 3200 [B through F]).

Arch Pathol Lab Med Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al 11

 Table 4. Common Immunohistochemical Stains
Used to Differentiate Primary Lung Carcinoma From
Carcinoma of Breast, Prostate, and Colorectal Origin
Lung Breast Prostatic Colorectal
Origin Origin Origin Origin
TTF-1a Mammaglobin PSA CDX2b
Napsin A GCDFP-15 PSAP
GATA3 NKX3.1
ERG
Abbreviations: ERG, v-ets erythroblastosis virus E26 oncogene homo-
logue; GCDFP-15, gross cystic disease fluid protein 15; PSA, prostate-
specific antigen; PSAP, prostate-specific acid phosphatase; TTF-1,
thyroid transcription factor 1.
a
TTF-1 may be positive in a subset of colorectal carcinomas.
b
CDX2 may be positive in mucinous adenocarcinoma of the lung.
studies have suggested that higher levels predict improved
survival.103,106 GATA3 may also stain background lympho-
cytes. Lastly, the use of this panel of immunostains can also
aid with the diagnosis in cytology specimens. Mammaglobin
and GCDFP-15 have high specificities for breast carcinomas
on cell block (88% and 96%, respectively), but have
suboptimal sensitivities (26% and 14%, respectively),
especially relative to that of GATA3 (86%).107,108 A
combined double stain of TTF-1 and napsin A has also
been proposed for these limited specimens, and it has been
demonstrated to be diagnostically useful in identifying
pulmonary adenocarcinomas on cell block.109
In addition to breast cancer, prostate and colorectal
cancers are 2 of the most common malignancies that lead
to pulmonary metastases. The common IHC stains used to
differentiate primary lung carcinoma from carcinomas of
breast, prostate, and colorectal origin are summarized in
Table 4. In the United States, prostatic adenocarcinoma is
the most common type of cancer in males and is estimated
to metastasize to the lungs in 46% of cases of metastatic
prostate cancer. In this setting, it is helpful to note that
metastatic prostate cancer rarely presents as an isolated
metastasis.110,111 Cytokeratin 7 can be used to differentiate
lung adenocarcinoma from prostatic acinar carcinoma (more
than 90% of which are negative for CK7 and cytokeratin 20
[CK20]), with the exception of prostatic ductal carcinoma,
which can be CK7 positive.101 Prostate-specific antigen and
prostate-specific acid phosphatase are sensitive and specific
cytoplasmic markers for identifying more than 90% of
metastatic prostate adenocarcinomas, although both may
demonstrate weaker expression in poorly differentiated
tumors.100,112 Although NK3 homeobox 1 (NKX3.1), a highly
sensitive nuclear marker for primary prostatic adenocarci-
noma, was previously thought to be lost in a majority of
metastatic disease, newer antibodies have demonstrated
near-100% sensitivity and specificity for high-grade and
metastatic prostate adenocarcinomas.112 Lastly, v-ets avian
erythroblastosis virus E26 oncogene homolog (ERG) im-
munostain has been recently described as a surrogate
marker for the TMPRESS2-ERG fusion transcript that is
reported in 40% to 70% of prostate cancer.113,114 Several
studies have shown ERG antibodies to be highly accurate in
identifying prostate cancers with ERG overexpression, in
both primary and metastatic disease.113–115
Colorectal cancer metastasizes to the lungs in 10% to 20%
of patients.116 CK7 and CK20 are frequently used as the
first-line markers to differentiate lung (CK7 positive, CK20
negative) from metastatic colorectal adenocarcinomas (CK7
12 Arch Pathol Lab Med
negative, CK20 positive).100 TTF-1 and napsin A can also be
included in the panel to support a lung primary, along with
caudal type homeobox 2 (CDX2), a highly sensitive nuclear
marker of intestinal adenocarcinomas, to support a colo-
rectal origin.117 However, caution should be taken when
interpreting this panel on mucinous adenocarcinoma of the
lung, as this subtype frequently shifts away from its lung
phenotype, and may be negative for TTF-1 and napsin A
and positive for CK20 and CDX2.100,118 Moreover, TTF-1 has
also been found to be positive in a subset of colorectal
carcinomas.11 In a study of 555 colorectal adenocarcinomas,
TTF-1 was positive in 18 (3.2%) and 24 cases (4.3%), using
the 8G7G3/1 and SPT24 clones, respectively.119
For other extrapulmonary adenocarcinomas, PAX8 has
also been recently proposed as a third immunomarker used
alongside TTF-1 and napsin A to distinguish primary lung
adenocarcinomas from metastatic neoplasms.120 PAX8 is a
nuclear protein that is negative in primary lung adenocar-
cinomas, and has positive expression restricted to epithelial
tumors of the thyroid, kidney, Müllerian system, and
thymus.121
In summary, to distinguish a primary pulmonary adeno-
carcinoma from a metastatic malignancy, an initial panel of
immunostains should include TTF-1, napsin A, CK7, and
CK20. Organ-specific markers should be added according to
clinical suspicion and morphologic impression. Breast
cancer markers include GATA3, mammaglobin, and
GCDFP-15. For prostate cancer, prostate-specific antigen
and prostate-specific acid phosphatase are established
markers capable of identifying more than 90% of metastatic
tumors. Newer markers for prostate cancer include NKX3.1,
as well as ERG, a highly specific marker in identifying
prostate tumors affected by a TMPRSS2-ERG gene fusion,
although it is a less sensitive marker overall. CDX2 may be
added to identify metastatic colorectal adenocarcinomas,
although caution should be taken when primary mucinous
adenocarcinomas of the lung is in the differential, as this
subtype may mimic the immunophenotype of colorectal
carcinoma. PAX8 offers high utility in identifying metastatic
adenocarcinomas to the lung, because it is negative in
primary lung adenocarcinoma but positive in epithelial
tumors of the thyroid, kidney, Müllerian system, and
thymus.
LUNG NEUROENDOCRINE TUMORS
A 72-year-old man with a 150–pack-year smoking history
presented with significant weight loss and CT chest imaging
that showed a right upper lobe lung mass and multiple
enlarged mediastinal lymph nodes. Microscopic examina-
tion of the endobronchial ultrasound biopsy revealed sheets
and nests of loosely cohesive, small, round to fusiform
hyperchromatic cells with finely granular chromatin, incon-
spicuous nucleoli, and scant cytoplasm (Figure 10, A and B).
There was prominent crush artifact. In better preserved
areas, the mitotic count ranged from 6 to 8 per high-power
field. The neoplastic cells were positive for CK7 (Figure 10,
C), synaptophysin (Figure 10, D), and TTF-1 (Figure 10, E);
they were negative for CK20 and chromogranin A. The Ki67
labeling index was greater than 90% (Figure 10, F). The final
diagnosis was SCLC.
With an incidence of more than 30 000 per year in the
United States, SCLC accounts for about 14% of all lung
cancers.122–125 Nearly all cases involve individuals with a
history of smoking.126 Only a minority of cases present with
Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al
Figure 10. Small cell lung carcinoma. The biopsy shows a sheet of small round hyperchromatic cells with peripheral crush artifact at low power (A).
At high power, the biopsy shows small round cells with scant cytoplasm, even and finely granular chromatin, inconspicuous nucleoli, and high
mitotic activity (B). The tumor cells are positive for cytokeratin 7 (C), synaptophysin (D), and thyroid transcription factor 1 (E). The Ki67 proliferative
index is greater than 90% of the tumor cells (F) (hematoxylin-eosin, original magnifications 340 [A] and 3400 [B]; original magnifications 3400 [D]
and 3100 [C, E, and F]).

Arch Pathol Lab Med Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al 13

 Table 5. Immunohistochemistry of Neuroendocrine Tumors of the Lung
Immunostain TC AC LCNEC SCLC
a
AE1/AE3 POS POS POS POS
CAM5.2 NEG/POS POS/NEG POS POS
CK7 NEG NEG POS NEG/POS
CK20 NEG NEG NEG NEG
CD56a POS POS POS/NEG POS
Synaptophysina POS POS POS POS/NEG
Chromogranin Aa POS POS POS/NEG NEG/POS
TTF-1a NEG NEG POS POS
Ki-67 (proliferation index)a 5% 20% 40%–80% 50%–100%
Abbreviations: AC, atypical carcinoid; CK, cytokeratin; LCNEC, large cell neuroendocrine carcinoma; NEG, negative; POS, positive; SCLC, small
cell lung carcinoma; TC, typical carcinoid; TTF-1, thyroid transcription factor 1.
a
Especially useful stains.

a small, localized lesion; most cases present with an neuroendocrine markers, a morphology highly characteristic
advanced stage and metastatic disease.126 Imaging studies of SCLC can support the diagnosis.128 Although TTF-1 can
can reveal a large mass demonstrating mediastinal invasion be positive in 70% to 90% of SCLCs,127,139–143 a study
or compression with regional lymphadenopathy; superior showed that 7 of 16 extrapulmonary small cell carcinomas
vena cava syndrome can occur in some instances.126 (44%) were also positive for TTF-1.144 Thus, TTF-1 has little
Small cell lung carcinoma can be easily recognized by its to no utility in determining the primary origin of the tumor.
distinct morphology.127,128 The neoplastic cells usually form A high Ki67 proliferation index can be very useful to
sheets and nests. Cytologically, they are small (less than 3 differentiate carcinoids from SLCL and LCNEC. Ki-67
times the diameter of a resting lymphocyte), hyperchro- proliferation index is usually up to 5% in typical carcinoid,
matic, and round to fusiform; they contain inconspicuous up to 20% in atypical carcinoid, 50% to 100% in SCLC, and
nucleoli, finely granular chromatin, and scant cyto- 40% to 80% in LCNEC.133 It should be noted, however, that
plasm.127,128 Mitotic figures may be difficult to identify on after chemotherapy, the high Ki-67 proliferation index of
small biopsies, but when present, they average about 80 SCLC can be reduced to levels more characteristic of
mitoses per 2 mm2.127,129,130 carcinoid tumors and thus be a source of confusion.128,145
Small cell lung carcinoma is one subclassification of The immunophenotypic profiles of SCLC and LCNEC are
neuroendocrine tumors of the lung, which also include highly similar. Thus, the distinction between the two
typical carcinoid, atypical carcinoid, and large cell neuroen- ultimately relies on morphologic examination. Small cell
docrine carcinoma (LCNEC). Typical carcinoid is character- lung carcinoma contains cells that are smaller (less than the
ized by a mitotic count of less than 2 per 2 mm2, atypical diameter of 3 small resting lymphocytes), have a higher
carcinoid is characterized by a mitotic count of 2 to 10 per 2 nuclear to cytoplasmic ratio, contain finely granular and
mm2, and high-grade neuroendocrine carcinomas (SCLC uniform chromatin, and have absent to inconspicuous
and LCNEC) are characterized by a mitotic count of more nucleoli, fusiform shape with scant cytoplasm, and crush
than 10 per 2 mm2. Necrosis is typically frequent, but it may artifact.128 In contrast, LCNEC is less uniform than SCLC
be missed because of limited sampling.128 Biopsy samples and contains polygonal-shaped cells with coarsely granular
may be subject to extensive crush artifact, which may limit to vesicular chromatin, prominent nucleoli, and abundant
the ability to render a definitive diagnosis. In these cases, pink cytoplasm. LCNEC is also less likely to demonstrate
cytology preparations may be better preserved and diag- nuclear molding.
nostic.128 In addition, in the larger resected specimens, Nonneuroendocrine mimickers of SCLC that are keratin
because of better fixation, the neoplastic cells tend to appear positive include Merkel cell carcinoma and various subtypes
bigger,131 which may pose confusion with LCNEC or other of SqCC (Table 6). Merkel cell carcinoma has a very similar
malignancies. immunophenotypic profile to that of SCLC; however,
In histologically challenging cases, especially in cases of
extensive crush artifact, a core panel of immunostains may
prove helpful to confirm the diagnosis: pan-cytokeratin
AE1/AE3 (AE1/AE3), CD56, chromogranin A, synaptophy- Table 6. Immunohistochemistry of Small Cell Lung
Carcinoma (SCLC) Versus Keratin-Positive Mimics
sin, TTF-1, and Ki-67 (Table 5).127,128,132,133
AE1/AE3 is useful to confirm an epithelial origin, because Immunostain SCLC MCC SqCC
a keratin-negative SCLC is extremely unusual.128 Although AE1/AE3 POS POS POS
CK7 is positive in about half of cases, CK20 is positive in less CD56 POS POS NEGa
than 10% of cases.134,135 Although CD56 is a highly sensitive Synaptophysin POS POS NEGa
marker (positive in 90%–100% of cases of SCLC),134,136,137 it Chromogranin A POS/NEG POS NEGa
TTF-1 POSa NEGa NEG
lacks specificity. Therefore, the diagnosis of SCLC in the CK20 NEGa POSa NEG
context of isolated CD56 positivity requires morphologic CK903 NEG ... POSa
correlation.128 Synaptophysin and chromogranin A are p63 NEG POS/NEG POSa
typically positive in SCLC, but in a study, no neuroendo- Abbreviations: CK, cytokeratin; MCC, Merkel cell carcinoma; NEG,
crine IHC activity was identified in 5 of 21 transbronchial negative; POS, positive; SqCC, squamous cell carcinoma; TTF-1,
biopsy specimens (24%) and 4 of 20 open lung biopsy thyroid transcription factor 1.
specimens (20%) of SCLC.138 In these cases negative for a
Especially useful results.
14 Arch Pathol Lab Med Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al
 Table 7. Immunohistochemistry of Small Cell Lung Carcinoma (SCLC) Versus Keratin-Negative Mimics
Immunostain SCLC Lymphoid Infiltrate/Lymphoma
AE1/AE3 POS NEG
CD45 NEG POS
CD99 NEG ...
S100 NEG ...
Abbreviations: NEG, negative; POS, positive.
although Merkel cell carcinoma is typically positive for CK20
(perinuclear dotlike pattern) and negative for TTF-1, SCLC
typically demonstrates the opposite profile (negative for
CK20 and positive for TTF-1).132,146 Although SqCC is
typically negative for neuroendocrine markers (CD56,
chromogranin A, and synaptophysin) and positive for
CK903 and p63, SCLC also typically demonstrates the
opposite profile (positive for CD56, CG, and synaptophysin
and negative for CK903 and p63).132,146,147
Nonneuroendocrine mimickers of SCLC that are keratin
negative include lymphoid infiltrates/lymphoma, Ewing
sarcoma, and melanoma (Table 7). CD45 can demonstrate
hematolymphoid differentiation. CD99 and S100 can help
screen for Ewing sarcoma and melanoma, respectively.
UNUSUAL LUNG NEOPLASMS
A 33-year-old woman with a history of familial adeno-
matous polyposis and abdominal desmoid tumor presented
with a heterogeneously enhancing 2.0 3 1.8-cm right upper
lobe nodule seen on chest CT. A CT-guided biopsy
demonstrated a low-grade epithelioid neoplasm (Figure
11, A) positive for AE1/AE3 (focally positive in rare scattered
cells) (Figure 11, B), Cam5.2 (Figure 11, C), and TTF-1
(Figure 11, D) and negative for CK7 (Figure 11, E). The
neoplastic cells were also negative for synaptophysin,
chromogranin, HMB-45, MART1, S100, and PAX8. The
histologic features and IHC staining results supported a final
diagnosis of sclerosing pneumocytoma.
Sclerosing pneumocytoma (formerly pulmonary scleros-
ing hemangioma) is an uncommon lung tumor arising from
primitive lung epithelium.148 It is typically seen in middle-
aged adults, with a female predilection (5:1 female to male
ratio).148 On imaging studies, sclerosing pneumocytoma
usually consists of a solitary (rarely multiple) nodule/mass
that is well defined, oval, and homogeneous.149
By histology, sclerosing pneumocytoma consists of 2
major cell types: surface cells and round cells. Surface cells
resemble reactive type II pneumocytes and are cuboidal,
whereas round cells consist of cells with well-defined
borders, fine chromatin, and inconspicuous nucleoli.150
Sclerosing pneumocytomas demonstrate 4 major patterns:
papillary, sclerotic, solid, and hemorrhagic, the former 3
patterns of which are the most common.148 Other histologic
Table 8. Immunohistochemical Differences
Between Surface Cells and Round Cells
of Sclerosing Pneumocytoma
Surface Cells Round Cells
TTF-1 TTF-1
EMA EMA
AE1/AE3
Abbreviations: EMA, epithelial membrane antigen; TTF-1, thyroid
transcription factor 1.
Arch Pathol Lab Med
Ewing Sarcoma Melanoma
NEG NEG
NEG NEG
POS ...
... POS
findings may include chronic inflammation, mast cells,
xanthomatous histiocytes, hemosiderin, calcifications, cho-
lesterol clefts, and large lamellar structures.148 Cytologic
atypia of the surface and round cells is usually not seen,
although moderate to marked atypia can occur on rare
occasions.150
The largest IHC study of sclerosing pneumocytoma
demonstrated that both surface and round cells stain for
TTF-1 and EMA.148 The surface cells are characteristically
positive for AE1/AE3 and also for CK7 and CAM 5.2. In
contrast, round cells are characteristically negative for AE1/
AE3, with only a few cases showing scattered cells positive
for CK7 and CAM 5.2 (Table 8). Antibodies for surfactant
proteins A and B are also positive in surface cells and
negative in round cells.148
The differential diagnosis of sclerosing pneumocytoma
comprises both benign and malignant entities (Table 9).
Differentiating sclerosing pneumocytoma from malignant
lung tumors (eg, adenocarcinoma and carcinoid tumor) and
metastatic carcinomas (eg, metastatic papillary thyroid
carcinoma and metastatic renal cell carcinoma) is especially
critical.
Distinguishing sclerosing pneumocytoma from lung
adenocarcinoma may be particularly challenging in cases
with subtle nuclear atypia. In this scenario, the presence of 2
distinct epithelial cell populations seen in the context of 1 of
the 4 classic patterns of sclerosing pneumocytoma may be
sufficient for a morphologic diagnosis. TTF-1 (staining both
surface and round cells) and AE1/AE3 (staining surface cells
only) can differentially highlight the 2 distinct epithelial cell
populations. Sclerosing pneumocytoma is negative for the
neuroendocrine markers chromogranin and synaptophysin.
Because sclerosing pneumocytoma can assume a papillary
configuration, a metastatic papillary thyroid carcinoma or
renal cell carcinoma may enter the differential diagnosis.
Although cytologic features seen in papillary thyroid
carcinoma can be helpful, immunostains for thyroglobulin
and/or PAX8 can also be used to exclude the possibility of a
metastatic papillary thyroid carcinoma. Similarly, the
marked cytologic atypia of a metastatic renal cell carcinoma
may favor a malignant process. Immunohistochemical
staining for CD10, carbonic anhydrase IX, and/or PAX8
(all typically positive in metastatic renal cell carcinoma) may
be helpful in these cases.
Benign tumors that enter the differential diagnosis of
sclerosing pneumocytoma include clear cell tumor, heman-
gioma, and pulmonary hamartoma. Clear cell tumors
typically demonstrate strong HMB-45 expression. Heman-
giomas typically stain positively for vascular markers such as
CD31, CD34, and ERG1. Pulmonary hamartoma, which
demonstrates varying degrees of mature cartilage, smooth
muscle, and/or adipose tissue, is usually diagnosed by
morphology alone. Immunohistochemical markers are
Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al 15
Figure 11. Sclerosing pneumocytoma. The biopsy shows a low-grade epithelial neoplasm (A) positive for AE1/AE3 (focal) (B), Cam5.2 (C), and
thyroid transcription factor 1 (D) and negative for cytokeratin 7 (E) (hematoxylin-eosin, original magnification 3400 [A]; original magnification 3400
[B through E]).

16 Arch Pathol Lab Med Immunohistochemistry in Lung and Pleural Neoplasms—Woo et al


Table 9. Differential Diagnosis of Sclerosing
Pneumocytoma
Differential Diagnosis Immunostain
Benign
Clear cell tumor HMB-45
Hemangioma Vascular markers (CD31,
CD34, ERG)
Hamartoma S100 (mature
chondrocytes)
Malignant
Adenocarcinoma in situ TTF-1
Carcinoid tumor Synaptophysin,
chromogranin A
Metastatic papillary Thyroglobulin, PAX8
thyroid carcinoma
Metastatic renal cell CD10, CA IX, PAX8
carcinoma
Abbreviations: CA IX, carbonic anhydrase IX; ERG, v-ets avian
erythroblastosis virus E26 oncogene homolog; PAX8, paired box gene
8; TTF-1, thyroid transcription factor 1.
generally not needed, but S100 may be used to highlight
mature chondrocytes.
CONCLUSIONS
In conclusion, the judicious use of IHC stains can be
helpful in the small-biopsy setting. In addition to helping
the pathologist further characterize the origin of the tumor
cells in diagnostically challenging biopsies, IHC has also
become increasingly important to identify diagnostic,
therapeutic, and prognostic biomarkers in cases of NSCLC.
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