AJCP / Original Article
Evaluation of Napsin A, TTF-1, p63, p40, and
CK5/6 Immunohistochemical Stains in Pulmonary
Neuroendocrine Tumors
Chen Zhang, MD, PhD,1 Lindsay A. Schmidt, MD,2 Kazuhito Hatanaka, MD, PhD,3
Dafydd Thomas, MD,2 Amir Lagstein, MD,2 and Jeffrey L. Myers, MD2
From the 1Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis; 2Department of Pathology, University
of Michigan, Ann Arbor; and 3Department of Molecular and Cellular Pathology, Kagoshima University Graduate School of Medical and Dental Sciences,
Kagoshima, Japan.
Key Words: Pulmonary neuroendocrine tumor; Carcinoid; Immunohistochemistry; Napsin A
Am J Clin Pathol September 2014;142:320-324
DOI: 10.1309/AJCPGA0IUA8BHQEZ
ABSTRACT
Objective: A panel of immunohistochemical (IHC) stains
frequently used to subclassify non–small cell lung cancers
(NSCLCs) includes napsin A, TTF-1, CK5/6, p40, and p63.
The expression profiles of these stains in neuroendocrine
tumors have not been systematically evaluated.
Method: Sixty-eight resected pulmonary neuroendocrine
tumors, including 52 typical carcinoids (TCs), eight atypical
carcinoids (ACs), seven small cell carcinomas (SCLCs)
and one large cell neuroendocrine carcinoma (LCNEC),
were stained for napsin A, TTF-1, p63, p40, and CK5/6.
Tumors were scored as positive (>1% tumor cells reactive)
or negative, and percentage of reactive tumor cells was
recorded.
Results: Napsin A, p63, p40, and CK5/6 were consistently
negative in neuroendocrine tumors. TTF-1 was positive in 17
of 52 TCs, 4 of 8 ACs, 5 of 7 SCLCs, and 0 of 1 LCNECs.
Conclusion: Pulmonary neuroendocrine tumors have a
distinct but nonspecific profile on IHC panel commonly
applied to subclassify NSCLCs. They are napsin A–/p40–/
p63–/CK5/6–/TTF-1±. Recognizing this profile may have
value in separating neuroendocrine tumors from NSCLCs.
320 Am J Clin Pathol 2014;142:320-324
320 DOI: 10.1309/AJCPGA0IUA8BHQEZ
Downloaded from https://academic.oup.com/ajcp/article-abstract/142/3/320/1760872 by guest on 20 February 2019
Pulmonary neuroendocrine tumors include a spectrum
of neoplasms from low-grade typical carcinoid tumors (TCs)
and intermediate-grade atypical carcinoid tumors (ACs) to
high-grade large cell neuroendocrine carcinoma (LCNEC)
and small cell carcinoma (SCLC),1 comprising 15% to 20%
of all pulmonary neoplasms.
Differentiating pulmonary neuroendocrine tumors
from other non–small cell lung cancers (NSCLCs)
including adenocarcinoma and squamous cell carcinoma
is critical because of significant differences in treatment
and prognosis. This differential diagnosis is usually not
difficult on resection specimens. However, with the advance
of radiology-assisted biopsy techniques and fine-needle
aspirations (FNAs), pathologic diagnosis of pulmonary
neoplasms is more commonly done preoperatively on small
biopsy specimens or FNA materials. In such specimens with
very limited numbers of tumor cells and minimal, absent,
or disrupted architecture, primary neuroendocrine lung
tumors may be confused with NSCLCs. For example, poorly
differentiated adenocarcinoma can form solid nests and
single cells only, without identifiable glandular architecture
or mucin production in a small biopsy, making it difficult to
differentiate from some neuroendocrine tumors.
Immunohistochemistry (IHC) can be helpful in
subclassifying lung carcinomas on small biopsies. An
increasing number of studies have focused on using various
antibodies to subclassify NSCLCs as either adenocarcinomas
or squamous cell carcinomas. Commonly used markers
include napsin A and TTF-1 for adenocarcinoma and CK5/6,
p63, and p40 for squamous cell carcinoma. The expression
profiles of these stains in neuroendocrine tumors have not
been systematically evaluated.
© American Society for Clinical Pathology
 AJCP / Original Article

Napsin A is an aspartic proteinase that is normally
expressed in type II pneumocytes. It is expressed in 65.3%
to 87% of pulmonary adenocarcinomas.2-6 Only a limited
number of cases of neuroendocrine tumors, including 16
TCs,4-6 one AC,5 and 86 SCLCs,2-6 have been studied and the
experience to date suggests that lack of napsin A expression
is characteristic of pulmonary neuroendocrine tumors. TTF-1
has been more extensively evaluated in lung neuroendocrine
tumors, but the results are variable.7-9 To our knowledge,
no previous study has examined p40 expression in lung
neuroendocrine tumors. Literature on the expression of p63
❚Table 1❚. Appropriate positive and negative controls were
used for all IHC studies. Positive IHC staining was defined
as staining in at least 1% of tumor cells. For TTF-1, p63, and
p40, only nuclear staining was recorded as positive. Each
case was scored as positive or negative, and the percentage of
positively staining tumor cells was recorded.
Results
IHC staining results are summarized in ❚Table 2❚ and

Downloaded from https://academic.oup.com/ajcp/article-abstract/142/3/320/1760872 by guest on 20 February 2019

and CK5/6 in lung neuroendocrine tumors is limited.10
We hypothesize that pulmonary neuroendocrine tumors
have a distinct but nonspecific profile using an IHC panel
commonly applied to small lung biopsies to subclassify
NSCLCs. Recognizing this profile may have value in
separating neuroendocrine tumors from other NSCLCs.
Materials and Methods
Case Selection
A combined total of 68 resected (including wedge
resections, lobectomy, and pneumonectomy specimens)
pulmonary neuroendocrine tumors over the past 5 years were
identified from the pathology archive. Institutional review
board permission was granted for the use of deidentified tissue
samples. H&E slides of all cases were reviewed and the cases
were divided into one of four categories using current World
Health Organization classification: 52 TCs, eight ACs, seven
SCLCs, and one LCNEC. The neuroendocrine nature of the
tumors was confirmed with IHC stains for synaptophysin and
chromogranin A. For TC, AC, and LCNEC, at least one of
these two markers was positive.
Immunohistochemistry
IHC staining for napsin A, TTF-1, p40, p63, and CK5/6
was performed on a single representative block from each case
using an automated immunostainer (Ventana-Biotech, Tucson,
AZ) and ultraView Universal DAB detection kit (Ventana-
Biotech). Antibody sources and dilutions are summarized in
representative napsin A staining as well as corresponding H&E
staining from different categories of neuroendocrine tumors
are shown in ❚Image 1❚. Napsin A, p63, p40, and CK5/6 were
consistently negative in all cases. Two neuroendocrine tumors
(one TC and one AC) showed focal immunoreactivity (<1%
of total tumor cells) for napsin A (Image 1B and Image D),
and were scored as negative. A review of the corresponding
H&E slides (Image 1A and Image C) showed that the
few scattered cells that were immunoreactive to napsin A
were morphologically different from the surrounding tumor
cells. They demonstrated lower nuclear/cytoplasmic ratio and
resembled entrapped pneumocytes or alveolar macrophages.
Variable TTF-1 immunoreactivity was observed in
different categories of neuroendocrine tumors. Seventeen of
52 TCs (ranging from 5%-80% of total tumor cells), four of
eight ACs (ranging from 5%-80% of total tumor cells), and
five of seven SCLCs (ranging from 80%-100% of total tumor
cells) were positive for TTF-1. The single case of LCNEC
was negative for TTF-1. TTF-1 staining in carcinoid tumors
was generally patchy and weak, compared with strong and
diffuse staining in SCLCs.
Discussion
Primary neuroendocrine lung neoplasms are consistently
negative for napsin A using a conventional immunostaining
technique. Napsin A was initially described in 1998 and is
a human aspartic proteinase related to pepsin, gastrin, renin,
and cathepsin.11 The highest levels of napsin A expression
are found in the lung, followed by the kidney, spleen, and

❚Table 1❚
Primary Antibodies Used in the Study

Antibody Clone Vendor/Location Dilution Digestion

Napsin-A IP64 Novocastra/Bannockburn, IL 1:100 None

TTF-1 8G7G3/1 Cellmarque/Rocklin, CA Predilute None
CK5/6 D5/16 Chemicon/Billerica, MA 1:100 HIER pH6
p63 4A4 Neomarkers/Fremont, CA 1:400 HIER pH6
p40 N/A BioCare Medical/Concord, CA 1:200 HIER pH6

© American Society for Clinical Pathology Am J Clin Pathol 2014;142:320-324 321

321 DOI: 10.1309/AJCPGA0IUA8BHQEZ 321
Zhang et al / Napsin A, TTF-1, p63, p40, and CK5/6 in Pulmonary Neuroendocrine Tumors

A B

Downloaded from https://academic.oup.com/ajcp/article-abstract/142/3/320/1760872 by guest on 20 February 2019

C D

E F

❚Image 1❚ H&E staining and corresponding representative napsin A staining in typical carcinoid tumor (A, B), atypical carcinoid
tumor (C, D), and small cell carcinoma (E, F) (×200).

322 Am J Clin Pathol 2014;142:320-324 © American Society for Clinical Pathology

322 DOI: 10.1309/AJCPGA0IUA8BHQEZ

❚Table 2❚
Napsin A, TTF-1, p63, p40, and CK5/6 IHC in Pulmonary Neuroendocrine Tumors
  No. of Positive Cases/No. of Total Cases (Range)
Napsin A TTF-1 p63 p40 CK5/6
TC 0/52 (0) 17/52 (5%-80%) 0/52 (0) 0/52 (0) 0/52 (0)
AC 0/8 (0) 4/8 (5%-80%) 0/8 (0) 0/8 (0) 0/8 (0)
SCLC 0/7 (0) 5/7 (80%-100%) 0/7 (0) 0/7 (0) 0/7 (0)
LCNEC 0/1 (0) 0/1 (0) 0/1 (0) 0/1 (0) 0/1 (0)
AC, atypical carcinoid tumor; IHC, immunohistochemistry; LCNEC, large cell neuroendocrine carcinoma; SCLC, small cell carcinoma; TC, typical carcinoid tumor.
leukocytes.12,13 In situ hybridization studies as well as IHC
studies have demonstrated napsin A expression in normal
human type II pneumocytes as well as in alveolar macrophages.7
Strong cytoplasmic staining of napsin A was seen in up to
87% of lung adenocarcinomas,2-6 and has been reported in
the surface component of sclerosing hemangiomas.14 The
sensitivity of napsin A IHC staining for diagnosing lung
adenocarcinoma is as good as, if not better than, TTF-1.
Napsin A expression in pulmonary neuroendocrine tumors
has been reported in only 17 cases of pulmonary carcinoids
in the English literature.4-6 Although the experiences from
these studies suggested that lack of napsin A expression is a
feature of pulmonary neuroendocrine tumors, ours is the first
study to systematically assess napsin A expression patterns
in primary neuroendocrine lung neoplasms. Interpretation
of napsin A staining can be complicated by staining in
entrapped pneumocytes and alveolar macrophages that may
be difficult to separate from neoplastic cells in small biopsy
specimens, making careful examination of corresponding
H&E-stained slides essential. Consistently negative staining
for napsin A in neuroendocrine tumors is particularly helpful
in separating them from adenocarcinomas that demonstrate
positive staining for neuroendocrine markers. Neuroendocrine
differentiation in lung adenocarcinomas is not uncommon
based on our own experience, and can be as high as 82%
in high-grade lung adenocarcinoma with fetal lung-like
morphology.15 IHC staining for neuroendocrine markers such
as synaptophysin and CD56 should not be used as a criterion
for establishing the diagnosis of neuroendocrine tumors in the
absence of supportive histologic and/or cytologic features.
TTF-1, a nuclear transcription factor that is expressed in
the developing forebrain, thyroid epithelium, and fetal lung
epithelial cells, is expressed in normal adult bronchiolar and
alveolar epithelium as well as many pulmonary neoplasms,
including most SCLCs and adenocarcinoma.8,16 In contrast to
the limited study of napsin A in carcinoids, TTF-1 expression
in carcinoids has been more extensively studied, and the results
are highly variable. The reported rates of TTF-1 positivity in
pulmonary carcinoids range from 0% to 69%, using the
same monoclonal 8G7G3/1 antibody.9,17,18 The wide range
of reported TTF-1 positivity in carcinoids may be partially
attributed to the case selection. For example, a high positive
© American Society for Clinical Pathology
AJCP / Original Article
rate of TTF-1 is seen in spindle cell carcinoid tumors.18
Downloaded from https://academic.oup.com/ajcp/article-abstract/142/3/320/1760872 by guest on 20 February 2019
Furthermore, the variation in the criteria for a positive stain in
these studies may be another factor contributing to the great
variation of results. In our current study, 33% of TCs and
50% of ACs demonstrated some degree of immunoreactivity
for TTF-1, which is consistent with the previously reported
study using similar scoring criteria.9 Although TTF-1
staining in carcinoid tumors was generally patchy and weak,
compared with its strong diffuse staining in SCLCs and
adenocarcinomas, there is great variation in staining intensity
in all categories of pulmonary neuroendocrine tumors and
adenocarcinomas, making it less useful in the differential
diagnosis of these tumors.
Carcinoid tumors are negative for p63, p40, and CK5/6.
p63 and CK5/6 are sensitive and relatively specific markers
of squamous differentiation. The majority of squamous
cell carcinomas are positive for both as demonstrated in
a recent study (n = 365; 90.3%), but a few express only
CK5/6 (n = 10; 2.2%) or p63 (n = 3; 0.7%).19 Polyclonal p40
antibody recognizes a p63 isoform, ΔNp63, which is a more
specific marker for squamous cell carcinoma, with sensitivity
comparable to that of p63.20 Most large-scale IHC studies of
p63, p40, and CK5/6 expression were done on squamous cell
carcinomas, with relatively fewer cases of adenocarcinoma
and SCLC. Patterns of expression for these markers in other
pulmonary neuroendocrine tumors such as TC and AC have
only rarely been reported.10 Pelosi et al21 recently showed
that two carcinoid tumors completely lacked p40 and p63 on
IHC staining, and that high-grade neuroendocrine tumors (10
SCLCs and five LCNECs) demonstrated very low IHC scores
for p40 and p63, especially p40. Our results were similar and
further support the observation that positive staining for any
of these markers is very uncommon and tends to mitigate
against the diagnosis of a pulmonary neuroendocrine tumor.
This can be especially helpful in cases of basaloid squamous
cell carcinoma, in which tumor cells may form peripheral
palisading structures that can be confused with the rosettes
seen in neuroendocrine tumors. In a recent study by Butnor
and Burchette,22 34 SCLCs were uniformly negative for p40,
whereas 12 (44.4%) of 27 biopsy samples of SCLC were
positive for p63. In our study with a relatively small sample
size, SCLC was negative for both p63 and p40.
Am J Clin Pathol 2014;142:320-324 323
323 DOI: 10.1309/AJCPGA0IUA8BHQEZ 323
Zhang et al / Napsin A, TTF-1, p63, p40, and CK5/6 in Pulmonary Neuroendocrine Tumors
The results of our study reveal a distinct but nonspecific
staining pattern of pulmonary neuroendocrine tumors using a
common IHC panel for NSCLCs. These tumors are consistently
negative for napsin A, which helps to differentiate them from
adenocarcinomas. CK5/6, p63, and p40 are equally effective in
differentiating squamous cell carcinoma from carcinoid tumors.
TTF-1 is not useful in the differential diagnosis between
pulmonary neuroendocrine tumors and adenocarcinomas.
Address reprint requests to Dr Myers: 2G332 UH, 1500 E Medical
Center Dr, Ann Arbor, MI 48109-0054.
References
1. Travis WD. Advances in neuroendocrine lung tumors. Ann
Oncol. 2010;21(suppl 7):65-71.
2. Turner BM, Cagle PT, Sainz IM, et al. Napsin A, a new
marker for lung adenocarcinoma, is complementary and more
sensitive and specific than thyroid transcription factor 1 in
the differential diagnosis of primary pulmonary carcinoma:
evaluation of 1674 cases by tissue microarray. Arch Pathol Lab
Med. 2012;136:163-171.
3. Stoll LM, Johnson MW, Gabrielson E, et al. The utility of
napsin-A in the identification of primary and metastatic lung
adenocarcinoma among cytologically poorly differentiated
carcinomas. Cancer Cytopathol. 2010;118:441-449.
4. Ueno T, Linder S, Elmberger G. Aspartic proteinase napsin is
a useful marker for diagnosis of primary lung adenocarcinoma.
Br J Cancer. 2003;88:1229-1233.
5. Bishop JA, Sharma R, Illei PB. Napsin A and thyroid
transcription factor-1 expression in carcinomas of the lung,
breast, pancreas, colon, kidney, thyroid, and malignant
mesothelioma. Hum Pathol. 2010;41:20-25.
6. Agackiran Y, Ozcan A, Akyurek N, et al. Desmoglein-3 and
napsin a double stain, a useful immunohistochemical marker
for differentiation of lung squamous cell carcinoma and
adenocarcinoma from other subtypes. Appl Immunohistochem
Mol Morphol. 2012;20:350-355.
7. Mori K, Kon Y, Konno A, et al. Cellular distribution of
napsin (kidney-derived aspartic protease-like protein, KAP)
mRNA in the kidney, lung and lymphatic organs of adult and
developing mice. Arch Histol Cytol. 2001;64:319-327.
8. Stahlman MT, Gray ME, Whitsett JA. Expression of thyroid
transcription factor-1(TTF-1) in fetal and neonatal human
lung. J Histochem Cytochem. 1996;44:673-678.
9. Srivastava A, Hornick JL. Immunohistochemical staining for
CDX-2, PDX-1, NESP-55, and TTF-1 can help distinguish
gastrointestinal carcinoid tumors from pancreatic endocrine
and pulmonary carcinoid tumors. Am J Surg Pathol.
2009;33:626-632.
324 Am J Clin Pathol 2014;142:320-324
324 DOI: 10.1309/AJCPGA0IUA8BHQEZ
10. Wang BY, Gil J, Kaufman D, et al. P63 in pulmonary
epithelium, pulmonary squamous neoplasms, and other
pulmonary tumors. Hum Pathol. 2002;33:921-926.
11. Tatnell PJ, Powell DJ, Hill J, et al. Napsins: new human
aspartic proteinases. Distinction between two closely related
genes. FEBS Lett. 1998;441:43-48.
12. Hirano T, Auer G, Maeda M, et al. Human tissue distribution
of TA02, which is homologous with a new type of aspartic
proteinase, napsin A. Jpn J Cancer Res. 2000;91:1015-1021.
13. Chuman Y, Bergman A, Ueno T, et al. Napsin A, a member
of the aspartic protease family, is abundantly expressed
in normal lung and kidney tissue and is expressed in lung
adenocarcinomas. FEBS Lett. 1999;462:129-134.
Downloaded from https://academic.oup.com/ajcp/article-abstract/142/3/320/1760872 by guest on 20 February 2019
14. Schmidt LA, Myers JL, McHugh JB. Napsin A is
differentially expressed in sclerosing hemangiomas of the
lung. Arch Pathol Lab Med. 2012;136:1580-1584.
15. Morita S, Yoshida A, Goto A, et al. High-grade lung
adenocarcinoma with fetal lung-like morphology:
clinicopathologic, immunohistochemical, and molecular
analyses of 17 cases. Am J Surg Pathol. 2013;37:924-932.
16. Folpe AL, Gown AM, Lamps LW, et al. Thyroid transcription
factor-1: immunohistochemical evaluation in pulmonary
neuroendocrine tumors. Mod Pathol. 1999;12:5-8.
17. Sturm N, Rossi G, Lantuejoul S, et al. Expression of thyroid
transcription factor-1 in the spectrum of neuroendocrine cell
lung proliferations with special interest in carcinoids. Hum
Pathol. 2002;33:175-182.
18. Tsuta K, Kalhor N, Wistuba II, et al. Clinicopathological
and immunohistochemical analysis of spindle-cell carcinoid
tumour of the lung. Histopathology. 2011;59:526-536.
19. Warth A, Muley T, Herpel E, et al. Large-scale comparative
analyses of immunomarkers for diagnostic subtyping
of non-small-cell lung cancer biopsies. Histopathology.
2012;61:1017-1025.
20. Bishop JA, Teruya-Feldstein J, Westra WH, et al. p40
(DeltaNp63) is superior to p63 for the diagnosis of pulmonary
squamous cell carcinoma. Mod Pathol. 2012;25:405-415.
21. Pelosi G, Rossi G, Cavazza A, et al. DeltaNp63 (p40)
distribution inside lung cancer: a driver biomarker approach
to tumor characterization. Int J Surg Pathol. 2013;21:229-239.
22. Butnor KJ, Burchette JL. p40 (DeltaNp63) and keratin
34betaE12 provide greater diagnostic accuracy than p63 in
the evaluation of small cell lung carcinoma in small biopsy
samples. Hum Pathol. 2013;44:1497-1486.
© American Society for Clinical Pathology