International Journal of Surgical Pathology
The online version of this article can be found at:
2013 21: 326 originally published online 5 June 2013 INT J SURG PATHOL
Giulio Rossi, Giuseppe Pelosi, Mattia Barbareschi, Paolo Graziano, Alberto Cavazza and Mauro Papotti
Small Cell Lung Cancer: Relevant Issues and Operative Recommendations for the Best
can be found at:
International Journal of Surgical Pathology
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International Journal of Surgical Pathology
21(4) 326 –336
© The Author(s) 2013
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The introduction of new effective chemotherapeutic agents
and targeted therapies in the management of non–small
cell lung cancer (NSCLC) has widened the spectrum of
therapeutic choices in the oncologists’ hands.1-14 However,
adoption of these new pharmaceutical agents requires pre-
definition of molecular features (ie, epidermal growth fac-
tor receptor [EGFR] mutations or anaplastic lymphoma
kinase [ALK] translocation)15,16 or precise subtyping of
NSCLC,17-20 at least dichotomizing squamous from non-
squamous cell carcinoma for clinical informed decisions
to take. In addition, recent investigations have suggested
that adenocarcinoma tends to metastasize to the lymph
nodes and central nervous system with a significantly
higher frequency than squamous cell carcinoma.21-23
Adenocarcinoma is also statistically associated with a bet-
ter median survival time when brain metastases are radio-
therapy treated or when radiotherapy is intended with a
curative intent in NSCLC.23
As highlighted for chemotherapy in the treatment of
lung cancer,24 even conventional morphology has reached
a plateau in NSCLC subtyping. The most careful examina-
tion of microscopic features on cytology and small biop-
sies leave a substantial rate of NSCLC not otherwise
specified (NOS; ranging from 10% to 30%), even in the
hands of expert pulmonary pathologists.25-28 This is mainly
because of the difficulty in translating all the morphologic
489346 IJSXXX10.1177/1066896913489346International Journal of Surgical PathologyPelosi et al
1Azienda Arcispedale S. Maria Nuova/IRCCS, Reggio Emilia, Italy
2Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy
3Università degli Studi, Milan, Italy
4San Camillo-Forlanini High Specialization Hospital, Malpighi Pavilion,
5Santa Chiara Hospital, Trento, Italy
6San Luigi Gonzaga Hospital and University of Torino/Orbassano, Italy
Giuseppe Pelosi, Dipartimento di Patologia Diagnostica e Laboratorio,
Fondazione IRCCS Istituto Nazionale Tumori, Via G. Venezian, 1I-
20133 Milan, Italy.
Subtyping Non–Small Cell Lung
Cancer: Relevant Issues and Operative
Recommendations for the Best Pathology
Giulio Rossi, MD1, Giuseppe Pelosi, MD, MIAC2,3, Mattia Barbareschi, MD4,
Paolo Graziano, MD5, Alberto Cavazza, MD1 and Mauro Papotti, MD6
Morphology still remains the cornerstone in lung cancer classification and cytology and small biopsy samples should
be interpreted by morphology, whenever feasible, according to shared and widely agreed-upon diagnostic schemes.
However, as novel therapy strategies are being offered on the basis of the diverse tumor characteristics, pathologists
are now challenged by the need to offer clinicians more detailed typing of non–small cell lung cancer, not otherwise
specified (NSCLC-NOS), especially when dealing with limited diagnostic material or poorly differentiated tumors. Close
integration of morphology, immunohistochemistry, and clinical data is highly warranted according to a multidisciplinary
approach to limit the category of NSCLC-NOS as much as possible or exclude unsuspected metastases, so rendering
more definite and clinically useful diagnoses. Among the many proposed immunohistochemical markers, which as a
whole are more practical and diagnostically useful than cumbersome and expensive molecular assays, a 2-hit model
including thyroid transcription factor-1 (TTF-1) and p40 (the latter more specific for squamous differentiation than
p63) seems to be the most effective to basically highlight adenocarcinoma (positivity for TTF-1 regardless of p63) and
squamous (always strongly and diffusely positive for p40 or p63 and negative for TTF-1) differentiation. This minimalist
2-hit diagnostic approach paves the way to novel perspectives in clinical trials on lung cancer, and it is also in keeping with
the need of strategically preserving diagnostic material for molecular assays that are essential for personalizing therapies.
non–small cell lung cancer (NSCLC), morphology, biopsy, cellblock, immunohistochemistry, TTF-1, p40, p63
Rossi et al
findings observed at a cytology/biopsy level, particularly
in poorly differentiated tumors,25-27 also because lung can-
cer classification has been constructed on resection speci-
mens and only recently the issues of cytology and small
biopsy specimens have been pointed out.27,29,30 Other
sources of inconsistency may derive from the inherent het-
erogeneity of adenocarcinoma and interobserver variabil-
ity because of the many histological subtypes and variants
existing,31 so the eventual subtyping of NSCLC may be
affected according to the axiom “the more the tumor cate-
gories, the more the difficulties in diagnosis.”32
In this frame of mind, immunohistochemistry (IHC)
has been representing the ancillary technique with the best
cost/benefit ratio in the distinction of NSCLC histo-
types,27,34 whereas molecular methods, namely micro-
RNA expression analysis, which have recently been
proposed, are unlikely to be directly transferred into the
everyday diagnostic workflow.35-38 Based on an extensive
literature review and our own experience, in June 2009 we
first suggested in this journal the adoption of an algorithm
comprising the best antibodies at that time able to identify
glandular (thyroid transcription factor-1 [TTF-1] and
CK7) and squamous cell (p63 and CK5/6) differentiation
in the setting of NSCLC.34 Needless to say that IHC is not
a perfect mathematic model, since there is a small subset
of NSCLC (hopefully <5%) with ambiguous co-expres-
sion of glandular and squamous cell differentiation mark-
ers or negative reaction for any marker, which an
unchanged eventual diagnosis of NSCLC-NOS may really
be justified for, as pointed out in the recent International
Association for the Study of Lung Cancer/European
Respiratory Society/American Thoracic Society (IASLC/
ERS/ATS) adenocarcinoma classification.29 Presently,
these tumor patients should undergo molecular assays for
EGFR mutations or ALK translocation or be candidates
for pemetrexed or bevacizumab therapy just like
This review was aimed at critically summarizing the
results of several published articles on NSCLC subtyping
in the past 3 years, issuing some practical key messages to
address the most limited panel of antibodies, including
recently introduced markers (ideally 1 marker for adeno-
carcinoma and 1 marker for squamous cell carcinoma),
compare older and newer diagnostic profiles, and provide
some operational rules to follow in the routine practice for
the most reliable diagnoses to render.
Morphologic Yield of Cytology and
Biopsy Specimen Examination
The rate of NSCLC-NOS was very high in the past when
there was no clinical reason for subtyping.26 A systematic
review by Paech et al39 evidenced that the agreement
between pathologists in discriminating squamous cell car-
cinoma from nonsquamous cell carcinoma on morphol-
ogy alone was moderate to high (k = 0.48-0.84). Rekhtman
et al40 were able to perform a definitive diagnosis of
NSCLC subtype in 169 out of 196 (88%) preoperative
cytology samples by morphologic criteria alone. In addi-
tion, a diagnosis of favored histologic type was made in
8%, then leaving unclassified NSCLC-NOS in 4% of
cases. Concordance between cytology and histology was
of 93%. In 165 tumors with surgical diagnosis of squa-
mous cell carcinoma or adenocarcinoma, preoperative
cytology had an accuracy of 96%, reaching 100% when
IHC stains were added. Nizzoli et al41 claimed that cytol-
ogy was able to subtype NSCLC in 85% of cases (158 out
of 183), whereas only 2% of NSCLC (3 cases out of 183)
were not classified at histology. The concordance between
cytology and histology was consistently high (88% of
cases, with k concordance value being 0.75). In both stud-
ies,40,41 squamous cell carcinoma histology had a lower
accuracy rate when compared with adenocarcinoma.
Comparing diagnosis on cytomorphology and surgical
resection specimens in 53 cases, Khayyata et al42 reported
66% accuracy for adenocarcinoma and 53% for squamous
cell carcinoma, with combined accuracy for either diagno-
sis being 60%. Sigel et al43 demonstrated that small biopsy
and cytology have a similar rate of accuracy in NSCLC
subtyping with a concordance value of 93% leading to a
NSCLC-NOS rate of 4%. This excellent result clearly
depends on the different expertise of pathologists in lung
cancer recognition as recently evidenced by Pelosi et al.44
The authors showed that the correct morphologic diagno-
ses on small biopsies of NSCLC arose from 67% to 84%
when expert pathologists reviewed slides.44 Finally, da
Cunha Santos et al45 confirmed the high agreement
between fine-needle aspiration cytology and histology on
surgical resections with an accuracy rate ranging from
96% to 85% in the diagnosis of adenocarcinoma and squa-
mous cell carcinoma, respectively. Of note, the percentage
of NSCLC-NOS significantly decreased from 35% to 24%
for the clinical purposes of targeted therapies (P = .01) to
reach 17% rate when exploiting IHC methods.45
Relevant points to morphology: At least 70% of lung
cancer patients are diagnosed in cytology and biopsy
specimens by using only light microscope without
attaining special stains.
What’s New From
Several articles have investigated the value of IHC in
NSCLC subtyping using different combinations/panel of
antibodies.46-73 Overall, there is agreement in limiting the
International Journal of Surgical Pathology 21(4)
use of immunostains to preserve as much tissue as possible
for molecular analyses, then performing 1 adenocarci-
noma and 1 squamous marker, as also recommended in the
new classification of adenocarcinoma.29,74
At the end, the coordinated expression for TTF-1 or
napsin-A for adenocarcinoma and p63 or p40 for squa-
mous cell carcinoma represents the most reasonable panel
in terms of sensitivity and specificity.27,29,30 Detailing on
few important articles, Downey et al53 found that all squa-
mous cell carcinomas (21 cases) were strongly positive
for CK5/6 and negative for TTF-1, whereas some adeno-
carcinomas (2 out of 24) stained with CK5/6. More
recently, Loo et al57 evidenced that Alcian blue/periodic
acid Schiff (AB/PAS) plus p63 and TTF-1 constituted the
best panel in subtyping NSCLC with 86% of diagnostic
accuracy. When prospectively applied to 82 biopsies, the
concordance between the immunohistochemical panel
results and morphologic subtype was complete (100%).
The category of NSCLC-NOS was then downrated to 7%.
Nicholson et al58 demonstrated a NSCLC refinement in
65% of cases using a panel of stains, including TTF-1,
p63, CK5/6, and PAS-diastase. The authors also under-
lined that high-molecular-weight cytokeratins cocktail
34βE12 showed consistent staining even in adenocarcino-
mas, then suggesting a limited role for this antibody.
Khayyata et al42 did not report immunoexpression for
TTF-1 in squamous cell carcinoma, whereas reactivity for
p63 and CK5/6 was not found in adenocarcinomas.
Pelosi et al44 matched the immunoprofiles of small biop-
sies and surgical resections of 63 cases using a panel of
markers comprising TTF-1, CK7, CK5/6, p63, and vimen-
tin. Revised morphology recognized 84% of corrected
cases, whereas 94% (59 out of 63) were correctly diag-
nosed on small biopsies using the aforementioned immuno-
panel. Squamous cell carcinomas did not stain for TTF-1,
whereas 9 out of 31 expressed CK7. One and 7 out of 22
adenocarcinomas showed some staining with CK5/6 and
p63, respectively. Hierarchic clustering analysis showed a
nonrandom distribution of immunoprofiles both in surgical
specimens and biopsies, this supporting the notion that
IHC-assessed small biopsies by using TTF1, CK5/6 and
CK7, p63, and vimentin could effectively parallel the cor-
responding profiling and hence eventual diagnoses on sur-
gical specimens. Mukhopadhyay and Katzenstein62 claimed
the usefulness of an antibody panel comprising TTF-1,
napsin-A, p63, and CK5/6 as the best combination in
NSCLC subtyping examining a series of 20 adenocarcino-
mas, 15 squamous cell carcinomas, and 4 large cell carci-
nomas. In their series, TTF-1 and napsin-A selectively
stained adenocarcinomas (80% and 58%, respectively) or
large cell carcinomas (50% and none, respectively), but
none of these markers was detected in squamous cell carci-
nomas. By contrast, p63 and CK5/6 stained 20% and 0% of
adenocarcinomas, respectively. Of interest, the final
suggested algorithm for NSCLC subclassification showed
that squamous cell carcinoma was favored only when dif-
fuse and strong immunostaining for p63 was observed in
absence of TTF-1 and/or napsin-A expression. Otherwise,
any staining for TTF-1 and/or napsin-A significantly fea-
tured adenocarcinoma. Since focal staining for p63 can be
observed in TTF-1 negative/napsin-A negative adenocarci-
nomas, but never in squamous cell carcinoma, a diagnosis
of poorly differentiated nonsquamous cell carcinoma seems
to be reasonable in this occurrence.62
Righi et al59 recently tested several antibodies, including
CK7, CK5, TTF-1, and p63 in a series of cellblocks from
103 fine-needle aspiration cytology samples. In addition,
p63, p40, napsin-A, and desmocollin-3 were tested when
discordant results were obtained using the first panel. In all
cases, a correlation with the relevant surgical specimens
was performed. Forty-six cases (44.7%) had a conventional
immunoprofile (TTF-1/CK7 positive and p63/CK5 nega-
tive profile for adenocarcinoma and the opposite for squa-
mous cell carcinomas). Fifty-seven cases had an ambiguous
phenotype. In these cases, TTF-1 had the strongest predic-
tive value in confirming adenocarcinoma, even when p63
was positive. Squamous cell carcinoma was favored when
tumors were strongly positive for p63 and negative for
TTF-1, whatever the immunostaining was for CK5 and
CK7. Again, adenocarcinoma was favored in the presence
of CK7 alone. At the end, only 14% of poorly differenti-
ated tumors remained NSCLC-NOS in this series of cyto-
logic samples. Of note, desmocollin-3 and p40 (the
truncated variant of p63) consistently confirmed squamous
cell carcinoma phenotype, whereas napsin-A was selec-
tively expressed in adenocarcinomas.59
In agreement with the majority of published works on
this topic and previous gene expression profiling studies,
Conde et al60 found that p63 expression was strikingly and
significantly associated with squamous cell carcinoma,
whereas 2 cases of NSCLC-NOS co-expressing TTF-1 and
p63 were considered to be squamous cell carcinoma.
Disowning the work by Conde et al,60 it is common opin-
ion, as also recently reported by Bishop et al,38 that NSCLC-
NOS co-expressing TTF-1 and p63 should be considered as
being poorly differentiated adenocarcinomas.
In the group of promising biomarkers to subtype
NSCLC, napsin-A for adenocarcinoma75-78 and p40 and
desmocollin-3 for squamous cell carcinoma have been
recently proposed.55,56,59,79-81 Of note, napsin-A seem to be
never expressed in squamous cell carcinoma and have
higher sensitivity than TTF-1 in poorly differentiated ade-
nocarcinoma. Desmocollin-3 has been recently proposed
by Monica et al82 on a series of large cell carcinomas,
which appears to have high specificity rate for squamous
cell carcinoma. Desmocollin-3 was never expressed in
adenocarcinoma and neuroendocrine tumors and was
mutually exclusive with TTF-1.81,82
Rossi et al
Despite a limited number of studies, p40 (an antibody
reacting only with truncated dominant-negative isoforms
of p63 gene, also named as deltaN-p63) seems to have an
higher specificity than conventional transactivated p63 in
distinguishing squamous cell carcinoma, and p63-positive
adenocarcinomas turned-out negative when using p40.64-67
Pelosi et al66 and Bishop et al67 robustly demonstrated that
p40 may be considered equivalent to p63 in terms of sen-
sitivity for squamous cell carcinoma, but it is significantly
superior in terms of specificity, since only rare adenocarci-
nomas may show p40 labeling in no more than 5% of
tumor cells if compared with p63 (3% vs 31%, respec-
tively).67 Therefore, we have recently recommended that
p40 may stably enter the diagnostic repertoire of every
pathologist to pave the way to a driver, holistic, biomarker
approach to lung cancer characterization for clinical pur-
poses, where a p40-based scoring system sharply sepa-
rated adenocarcinoma from squamous carcinoma
according to the axiom “no p40, no squamous.”81 At vari-
ance with TTF1, however, p40 was diagnostically power-
ful and meaningful when looking at either positive or
negative profiles, whereas TTF1 was diagnostically useful
especially if positive, since whenever negative in the set-
ting of poorly differentiated tumors could underline
diverse tumor subtypes, such as squamous cell carcinoma,
adenocarcinoma, adenosquamous carcinoma, sarcomatoid
carcinoma, and even salivary gland–type tumors.66,81
Finally, an old and apparently useless marker, namely
vimentin, has recently been reappraised for diagnosing
challenging NSCLC subtypes, such as sarcomatoid carci-
noma, more reliably, even on small biopsy samples,
whether a strong and diffuse cytoplasmic immunoreactiv-
ity was seen in most tumor cells.44,82
Nuclear markers are more reliably and easily interpre-
table than cytoplasmic markers especially when dealing
with limited diagnostic material, crush artifacts, and pas-
sive diffusion of cytoplasmic proteins. Although mixing
diverse biomarkers, whether nuclear or cytoplasmic, into
different antibody cocktails (eg, TTF-1/desmocollin-3,
p63/napsin-A, or p40/TTF-1) could be an adequate answer
in term of sensitivity and specificity of reaction to get
valuable diagnostic results,33,59 these combinations how-
ever need further evaluation.
The distribution of the main markers thus far proposed
for lung cancer subtyping and the relevant combinations
aimed at addressing the main differential diagnoses are
shown in Tables 1 and 2, respectively. Representative pic-
tures featuring diagnostic profiles according to TTF-1,
napsin-A, p63, and chromogranin immunoreactivity are
shown in Figure 1.
Relevant points to immunohistochemistry: A limited
use of IHC markers is recommended for NSCLC-
NOS to subtype. The best single marker for
adenocarcinoma is TTF-1 and the best single marker
for squamous cell carcinoma is p40, realizing a
2-hit, sparing material algorithm suitable for both
cytology and small biopsy specimens.
Be Aware of Antibodies Clones and
Other Technical Issues
When using immunohistochemistry for NSCLC subtyp-
ing, pathologists should be aware of some technical tricks
that can cause discordant or ambiguous results to occur. In
Table 1. Distribution of Immunomarker Expression Along the
3 Main Cell Differentiation Lineages of Epithelial Lung Tumors.
ADSQC NE tumors
~60% to 100%a
~70% to 100%
~20% to 30%
~20% to 30%
60% to 80%
~70% to 90%
Abbreviations: AD, adenocarcinoma; SQC, squamous cell carcinoma;
NE, neuroendocrine; TTF-1, thyroid transcription factor-1; CK,
cytokeratin; HMWCK, high-molecular-weight cytokeratin cocktail (as
aSome primary pulmonary mucin-rich adenocarcinomas may express
and/or coexpress CK20 and CDX2 rather than CK7 and TTF-1. AD
stands for adenocarcinoma
bAbsent to variably present (especially in small cell carcinoma [SCLC])
Table 2. Immunostains in the Most Common Differential
Diagnoses of Lung Cancera.
aBasic rules: Napsin-A expression excludes squamous cell carcinoma
and small cell carcinoma; p40 expression excludes small cell carcinoma
and adenocarcinoma; thyroid transcription factor-1 (TTF-1) expression
excludes squamous cell carcinoma.
International Journal of Surgical Pathology 21(4)
particular, Matoso et al83 evidenced that some commer-
cially available antibodies recognizing TTF-1 may show
different results. The monoclonal antibody 8G7G3/1
seems to be more specific than the clone SPT24, this latter
staining several pulmonary non–small cell carcinomas of
all histotypes. By contrast, the majority of published
works revealed that the monoclonal antibody 8G7G3/1 is
quite specific for adenocarcinoma and neuroendocrine
tumors, whereas squamous cell carcinomas are negative.
Some caveats should be taken into account when using
TTF-1, since different papers have observed a consistent
expression also in nonpulmonary adenocarcinomas,
mainly from the gynecologic tract (particularly from
endometrium) and breast.84,85 Another important concern
is the dilution at which TTF-1 antibody is employed. In
our own experience it is preferable to adopt a dilution
higher than 1:500, since immunostaining is well main-
tained preventing nonspecific results, such as cytoplasmic
positivity or background staining (Pelosi G, personal
Two recent works by Ordonez86,87 have consistently
demonstrated that squamous cell carcinomas of the lung
do not exhibit napsin-A and TTF-1 positivity. Of note, the
same author underlines the need to be aware when evaluat-
ing reactivity of napsin-A and TTF-1, since some apparent
discrepant results in squamous cell carcinoma are related
to an erroneous interpretation of the strong expression of
these markers in tumor-entrapped hyperplastic type II
pneumocytes. The strong reactivity of napsin-A in intra-
alveolar macrophages should also alert, particularly in
small biopsies, cytology, or even tissue microarrays.86,87
Also, it is important to note that monoclonal napsin-A is
Figure 1. The distribution of chromogranin (Chr), thyroid transcription factor-1 (TTF-1), napsin-A (Naps), and p63
immunoreactivity is shown as a function of the 3 main subtypes of lung cancer, namely, squamous cell carcinoma (Chr−/TTF-1−/
Naps−/p63+), adenocarcinoma (Chr−/TTF-1+/Naps+/p63±), and small cell carcinoma (Chr±/TTF-1+/Naps−/p63−).
Rossi et al
less sensitive, but more specific than polyclonal napsin-A
antiserum, whenever this antibody is used in discriminat-
ing lung origin of adenocarcinomas.75
Currently, the single best marker of squamous cell car-
cinoma is a polyclonal rabbit antiserum against p40,62,63
which works well in paraffin sections with strong specific
nuclear decoration and no significant background stain-
ing if the antibody is particularly diluted (at least
1:2000/1:3000), made react with short incubation and
developed with a highly sensitive detection system to
avoid unspecific cytoplasmic background or inconsistent
nuclear decoration.44,66,81 Worth noting is that nonneoplas-
tic lung tissue always is devoid of p40 nuclear staining
apart from the basal cell layer of the bronchial epithelium,
with no specific nuclear staining in type II pneumocytes
Relevant points to technical issues: The accurate choice
of the reagent types and the staining procedures are
particularly crucial in the IHC characterization of
lung cancer because false results of staining, whether
positive or negative, may seriously affect the obtain-
ing of reliable final results.
MicroRNA (miRNA) are short noncoding genes playing a
regulatory role in gene expression. These biomarkers are
essential in tissue differentiation and some miRNA are tis-
sue specific.35 Lebanony et al36 recently demonstrated that
expression of miRNA-205 by quantitative real-time poly-
merase chain reaction on formalin-fixed, paraffin-embed-
ded samples of NSCLC were significantly higher in
squamous cell carcinomas than in adenocarcinomas,
reaching a sensitivity of 96% and specificity of 90%.
Similar results were subsequently observed by Del
Vescovo et al37 and Bishop et al38 confirming high sensi-
tivity and specificity of miRNA levels in distinguishing
squamous cell carcinoma from adenocarcinoma, even if
with a few notes of caution.88 In fact, miRNA-205 expres-
sion parallel results obtained by conventional morphology
and/or immunohistochemistry. Considering the high cost
and the laboratory equipment required for miRNA-205
determinations, the expression of miRNAs represents an
accurate diagnostic adjunct rather than a true improvement
over more conventional characterization of NSCLC.89
Relevant points to miRNA assessment: Although
miRNA assessment is appealing in lung cancer for
its biological implications, morphology and a lim-
ited number of IHC markers still remain the best
choice to efficiently subtype NSCLC.
Practical Hints for Surgical
As recently underlined by the IASLC/ATS/ERS recom-
mendations, knowledge of clinical data may be essential
in lung cancer diagnosis, such as smoking habit, tumor
location and escavation, imaging appearance or labora-
tory tests, and history of previous malignancies.29
Pathologists should be aware that it is not infrequent to
deal with primary lung adenocarcinomas that may
express some markers commonly considered for extra-
pulmonary differentiation and vice versa, such as CDX-2
and enteric differentiation.90,91
Pathologists should always consider the possibility to
share difficult cases with other colleagues and more
expert pathologists. This is good practice behavior, since
it does not cause delay in patient management and obvi-
ates the need for complicated diagnoses due to exagger-
ated IHC that not necessarily provide an advantage over
routine light microscopy, according to the axiom “the
more the immunostains, the more complicated the
Pathologists should also consider that a minority of
NSCLC (hopefully <5%) remains hard to be subtyped on
cytology and small biopsies. In addition, it is paramount to
put great consideration on proper tissue handling in order
to optimize therapeutic strategies. Once morphology and a
couple of immunostains have failed in NSCLC subtyping,
remaining tumor cells should be tested for molecular anal-
yses, in particular EGFR and ALK testing.
Of note, TTF-1 and p63 seem to represent the most
widely used and tested panel of antibodies in NSCLC sub-
typing. Unfortunately, these markers are not completely
mutually exclusive and ambiguous results showing double
positivity or negativity or other combinations (ie, focal
p63 positivity in absence of TTF-1 staining) may occur.
On the premise that we are still waiting for perfect immu-
nomarkers, if any, the adoption of some basic and easy-to-
use rules are necessary and quite helpful when approaching
TTF-1 and p63 to discriminate adenocarcinoma from
squamous cell carcinoma (Figure 2).
Basically, all poorly differentiated NSCLC (or alter-
natively, NSCLC-NOS) with some TTF-1 positivity
should be equated to poorly differentiated adenocarcino-
mas whatever results are observed with p63. NSCLC
showing double negativity or only focal immunostaining
for p63 in absence of TTF-1 should be considered as
poorly differentiated nonsquamous cell carcinomas,
keeping in mind that this immunophenotype can charac-
terize poorly differentiated adenocarcinoma, sarcomatoid
carcinomas, NE tumors and even metastatic tumors to the
lungs. Poorly differentiated squamous cell carcinomas
are instead evidenced by strong and diffuse p63
International Journal of Surgical Pathology 21(4)
expression and TTF-1 negativity, hence negativity for
p63 exclude by definition this tumor type. Nevertheless,
napsin-A for adenocarcinoma and p40, CK5/6 or desmo-
collin-3 for squamous cell carcinoma seem to represent
further alternatives, even though the reason for using
minimal stains is to spare tissue for molecular studies. To
this regard, the doublet TTF-1 and p40 is currently
deemed the best combination for typing NSCLC in every
type of material (Figure 3).27,66,80
Relevant points to surgical pathologists: A multidisci-
plinary approach also including imaging and clinical
information has been recommended applying to the
diagnosis-making process of lung cancer especially
in cytology and small sized biopsy specimens, as
well as using a minimalist approach by mean a cou-
ple of well-defined and specific biomarkers (eg,
TTF-1 and p40) whenever light microscope or mucin
stain fails to give ultimate answers.
Conclusive Remarks for the Best
Diagnosis of lung cancer, according to the recent
IASLC/ATS/ERS recommendations, is often a
complex puzzle requiring step-wise integration of
clinical and radiologic data with pathologic features
(morphology and immunohistochemistry).
NSCLC subtyping is a key point in tailoring novel
treatments and guiding molecular investigations.
Such a diagnosis can be performed on morphology
in about 2/3 of NSCLC, but 30% of NSCLC require
ancillary techniques (basically immunohistochem-
istry) because of scarce material, artifacts, or poor
TTF-1 plus p40 is the best panel to limit the need
for further immunostains and preserving tissue for
molecular tests. Napsin-A for adenocarcinoma and
CK5/6 and desmocollin-3 for squamous cell
Figure 2. The diagnostic categories resulting from non–small cell lung cancer, not otherwise specified (NSCLC-NOS) after typing
by immunohistochemistry according to thyroid transcription factor-1 (TTF-1) and p63 are shown: NSCLC, favor adenocarcinoma
(TTF-1+/p63±), NSCLC, favor squamous cell carcinoma (TTF-1−/p63+), and NSCLC-NOS (TTF-1−/p63±), the latter profile including
poorly differentiated adenocarcinoma, sarcomatoid carcinoma, neuroendocrine (NE) tumors, large cell carcinoma (only on surgical
specimens), or metastatic tumors.
Rossi et al
carcinoma are the best choices if further stains are
necessary. Other markers (CK7, SOX2, clone
34βE12) seem to be nonspecific.
Finally, in case of a clear-cut morphology, IHC
should be used only to exclude metastatic extrapul-
monary adenocarcinoma. In general, the more
stains you demand the more complicated become
the case, considering that some overt pulmonary
adenocarcinomas may express puzzling combina-
tions of diverse markers. Suffice it to say that TTF-1
is the “queen” and p40 is the “king” when opera-
tively used according to appropriate diagnostic
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
The author(s) received no financial support for the research,
authorship, and/or publication of this article.
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Figure 3. A comparison between p63 and p40 as identifiers of adenocarcinoma (TTF-1±/p63±/p40−), squamous cell carcinoma
(TTF-1−/p63+/p40+), and NSCLC-NOS (TTF1−/p63±/p40−) is shown.
Abbreviations: TTF-1, thyroid transcription factor-1; NSCLC-NOS, non–small cell lung cancer, not otherwise specified.
International Journal of Surgical Pathology 21(4)
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