ORIGINAL ARTICLE

Refining the Diagnosis and EGFR Status of Non-small Cell

Lung Carcinoma in Biopsy and Cytologic Material, Using a
Panel of Mucin Staining, TTF-1, Cytokeratin 5/6, and P63,
and EGFR Mutation Analysis
Andrew G. Nicholson, DM, FRCPath,* David Gonzalez, PhD, Pallav Shah, MRCP,
Matthew J. Pynegar, BSc,* Manjiri Deshmukh, FRCPath,* Alexandra Rice, FRCPath,*
and Sanjay Popat, MBBS, MRCP, PhD

Introduction: The dichotomization of non-small cell carcinoma

(NSCLC) subtype into squamous (SQCC) and adenocarcinoma
(ADC) has become important in recent years and is increasingly
required with regard to management. The aim of this study was to
determine the utility of a panel of commercially available antibodies
in refining the diagnosis on small biopsies and also to determine
whether cytologic material is suitable for somatic EGFR genotyping
in a prospectively analyzed series of patients undergoing investigation for suspected lung cancer.
Methods: Thirty-two consecutive cases of NSCLC were first tested
using a panel comprising cytokeratin 5/6, P63, thyroid transcription
factor-1, 34E12, and a D-PAS stain for mucin, to determine their
value in refining diagnosis of NSCLC. After this test phase, two
further pathologists independently reviewed the cases using a refined panel that excluded 34E12 because of its low specificity for
SQCC, and refinement of diagnosis and concordance were assessed.
Ten cases of ADC, including eight derived from cytologic samples,
were sent for EGFR mutation analysis.
Results: There was refinement of diagnosis in 65% of cases of
NSCLC to either SQCC or ADC in the test phase. This included 10
of 13 cases where cell pellets had been prepared from transbronchial
needle aspirates. Validation by two further pathologists with varying
expertise in lung pathology confirmed increased refinement and
concordance of diagnosis. All samples were adequate for analysis,
and they all showed a wild-type EGFR genotype.
Conclusion: A panel comprising cytokeratin 5/6, P63, thyroid
transcription factor-1, and a D-PAS stain for mucin increases diagnostic accuracy and agreement between pathologists when faced
*Department of Histopathology, Royal Brompton Hospital, London; National Heart and Lung Institute, Imperial College London, United Kingdom; Department of Molecular Diagnostics, The Institute of Cancer
Research, Surrey; Department of Respiratory Medicine, Royal Brompton Hospital; and Department of Medical Oncology, Royal Marsden
Hospital, London.
Disclosure: The authors declare no conflicts of interest.
Address for Correspondence: Andrew G. Nicholson, DM, FRCPath, Department of Histopathology, Royal Brompton Hospital, Sydney St, London
SW3 6NP, United Kingdom. E-mail: a.nicholson@rbht.nhs.uk
Copyright 2010 by the International Association for the Study of Lung
Cancer
ISSN: 1556-0864/10/0504-0436

436

with refining a diagnosis of NSCLC to SQCC or ADC. These small

samples, even cell pellets derived from transbronchial needle aspirates, seem to be adequate for EGFR mutation analysis.
Key Words: Lung, Cancer, EGFR, Immunohistochemistry, Transbronchial needle aspirate, Cytology, Non-small cell carcinoma,
Adenocarcinoma, Squamous cell carcinoma.
(J Thorac Oncol. 2010;5: 436441)

ntil recently, subclassification of non-small cell lung

cancer (NSCLC) into different histologic types had little
clinical significance, because treatments did not differ between the variants. However, several drugs have become
available in recent years with indication contingent on precise
definition of tumor subtype, in relation to both efficacy and
safety. Thus, the multitargeted antifolate pemetrexed is contraindicated in squamous cell carcinomas (SQCC) with several trials demonstrating no significant efficacy in this
NSCLC subtype,13 but superior efficacy in the adenocarcinoma (ADC) and large cell subsets over standard platinumdoublet chemotherapy. Bevacizumab development was discontinued in patients with squamous cell tumors due to an
over-representation of fatal pulmonary hemorrhage in this
subgroup,4 and squamous histology is now a contraindication
for this drug. The EGFR-directed tyrosine kinase inhibitors
erlotinib and gefitinib have superior efficacy in patients harboring somatic EGFR mutation, which is most frequently
observed in ADCs.5 Moreover, histology subtype may also
offer prognostic information.5 In addition to the above need,
to better identify histologic NSCLC subtypes, biopsies of
adequate size are also required for somatic genotyping. Recent trial data have demonstrated superiority of gefitinib over
cytotoxic chemotherapy in patients with NSCLC harboring
somatic EGFR mutation; whereas in those with wild-type
EGFR, first-line gefitinib is markedly detrimental compared
with chemotherapy. Thus, EGFR genotyping is now indicated
if considering therapy with first-line gefitinib.6
The NSCLC diagnostic paradigm is further compounded by the advent of transbronchial needle aspiration
Journal of Thoracic Oncology Volume 5, Number 4, April 2010

Journal of Thoracic Oncology Volume 5, Number 4, April 2010

(TBNA)/endobronchial ultrasound (EBUS) and endoscopic

ultrasound (EUS) guided aspiration, which mean that many
patients are being diagnosed and staged in a single procedure
without recourse to any form of biopsy.7 This combination of
events is therefore potentially problematic as the volume of
diagnostic tissue is becoming ever smaller, with greater
requirement for refinement of diagnosis.
A potential facilitator in subdividing NSCLCs in small
biopsies is immunohistochemistry. Although most data are
retrospective and based on resection or tissue microarray
specimens and not small biopsies, antibodies to epitopes such
as P63,8 10 cytokeratin (CK) 5/6,9,11 and 34E1212,13 are
known to stain selectively SQCCs and similarly thyroid
transcription factor-1 (TTF-1) for lung ADCs.9,10,14,15 We
therefore aimed to determine the utility of a panel of commercially available antibodies to refine the diagnosis of
NSCLC by immunohistochemistry, and also to determine
whether cytologic material is suitable for somatic EGFR
genotyping in a prospectively analyzed series of patients
undergoing investigation for suspected lung cancer.

Refining the Diagnosis and EGFR Status of NSCLC

along with the ancillary data, by the same pathologist

(A.G.N.), and a final diagnosis was made.
Based on data from the earlier test panel by A.G.N.,
two further pathologists (one thoracic specialist [A.R.] and
one general pathologist [M.D.]) independently assessed the
cases in similar fashion, and then reassigned their diagnoses
using a validation panel of TTF-1, P63, CK 5/6, and a D-PAS
stain for mucin.
Ten cases diagnosed as ADC, eight derived from cell
pellets, were then sent for molecular analysis to look for
evidence of EGFR mutations, where samples were available.
Genomic DNA was extracted from formalin-fixed, paraffinembedded tissue sections (five sections 10 m width for each
sample) using the QIAamp DNA formalin-fixed, paraffinembedded kit (Qiagen, UK). EGFR mutation analysis was
carried out using the EGFR29 Mutation Kit (DxS Ltd, Manchester, United Kingdom) according to manufacturers instructions.
This method can detect the 29 most common EGFR mutations
in exons 18 to 21, and the kit can detect 1% of mutant DNA in
a background of wild-type genomic DNA. Positive controls
were included in every run for the most common EGFR mutations and all of them gave positive results.

MATERIALS AND METHODS

Patients referred to the Royal Brompton hospital for
suspected lung cancer, whose biopsy procedure was confirmative, were included in the study. The methodology for
bronchoscopy in patients undergoing TBNA has been described previously.7 Samples obtained from EBUS and EUS
were also collected into saline. A separate container was used
for each lymph node station and processed as follows: aspirated material was placed in saline, one container per station.
Blood was removed via density gradient centrifugation using
Lymphoprep (Axis-Shield PoC AS), during which specimens
were spun at 2000 rpm for 20 minutes, followed by cytospin
preparation, and up to four slides were reviewed per station.
In positive cases, the remaining material was then placed in
10% formaldehyde and subsequently spun into a cell pellet
for routine histologic processing. As a test phase, all samples
were first reviewed by one pathologist (A.G.N.) and were
diagnosed as NSCLC-not otherwise specified (NSCLCNOS), SQCC, ADC, or small cell lung carcinoma (SCLC).
Diagnosis of SQCC and ADC was based on identifying the
criteria for diagnosis of these tumor types in the 2004 World
Health Organisation (WHO) classification, noting the caveat
that these criteria are based on resection specimens.16 These
were the presence of keratinisation and/or intercellular digitation for SQCC and the presence of lepidic/acinar/papillary
architectures for ADC.
Positive biopsies and positive cell pellets were then
subjected to an ancillary panel of immunohistochemistry for
TTF-1 (Dako M3575, 1:100 dilution), p63 (Dako M7247,
1:2000), CK 5/6 (Dako M7237, 1:200), 34E12 (Dako
M0630, 1:150), with additional staining for broad spectrum
CKs using MNF116 (Dako M0821, 1:50), and neuroendocrine differentiation using CD56 (Vector VP-C360, 1:100)
when appropriate. Staining for neutral mucin using a diastase-resistant periodic acid Schiff (D-PAS) stain was also
undertaken. Each specimen was then reviewed a second time,

RESULTS
Between May 2008 and March 2009, 38 consecutive
biopsies were confirmed as primary lung cancer by one
pathologist (A.G.N.) in patients attending the Royal Brompton Hospital and Royal Marsden Hospital multidisciplinary
Thoracic Oncology service. Six of these were diagnosed as
SCLC, all confirmed by positive staining with neuroendocrine markers. Of the remaining 32 NSCLC cases, 12 were
endobronchial biopsies, five were core biopsies (lung 4,
liver 1), 13 were TBNAs (blind, EBUS, and EUS guided)
with subsequent cell pellets, and two were pleural effusions
with subsequent cell pellets.
Seven of 32 cases were diagnosed as ADC, of which
62.5% stained with TTF-1, with negative staining for CK5/6
and only one case showing focal staining with P63, whereas
50% stained with 34E12. Eight of 32 cases were diagnosed
as SQCC, all of these being negative TTF-1 and all positive
for CK5/6, P63, and 34E12. One of these showed very
occasional staining for mucin but was still classified as SQCC
as the tumor cells fulfilled WHO morphologic criteria for a
diagnosis of SQCC. The remaining 17 cases were diagnosed
as NSCLC-NOS, with 10 of the 13 TBNA/cell pellet specimens being within this group.
Subsequent to the primary immunohistochemistry
panel, a diagnosis of NSCLC-NOS was refined to ADC in 9
of 17 patients, tumor cells being either TTF-1 positive (9 of
9) and/or D-PAS mucin positive (3 of 8). These cases were
either wholly negative or only focally positive for CK5/6
(2 of 10) and P63 (2 of 10) (Figure 1), although there was
a greater degree and extent of positivity for 34E12 (9 of
9). Three of these cases were noted to have marked nuclear
pleomorphism. In two patients, the diagnosis of NSCLCNOS was refined to SQCC, these both being positive for
CK 5/6, P63, and 34E12 and negative for both TTF-1 and

Copyright 2010 by the International Association for the Study of Lung Cancer

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Nicholson et al.

mucin expression, although one case was only focally

positive for CK5/6.
In six cases, the final diagnosis remained as NSCLCNOS, four of which were cell pellets derived from TBNAs.
This was due to a lack of additional tumor cells within the cell
pellet to facilitate immunohistochemistry in two cases, an
inability to distinguish confidently between contaminating
native benign respiratory epithelial cells and tumor cells
showing lesser degrees of cellular atypia in two cases, and no
staining other than for 34E12 in two cases.
Therefore, 34E12 was dropped from the validation
panel, because the results proved noncontributory to diagnosis because of frequent staining of both ADCs and SQCCs.
After this prospective test phase, a second retrospective
independent review by two further pathologists (A.R. and
M.D.), blinded to the initial data, was undertaken on all 38
samples. On routine staining, there was agreement between
these two pathologists in 31 cases, classified by both as SCLC
(n 6), ADC (n 7), SQCC (n 4), and NSCLC-NOS (n
14). Seven of the remaining cases had discordant classifications,
with four being called SQCC by one pathologist and NSCLCNOS by the other (SQCC/NSCLC), and three ADC by one
pathologist and NSCLC-NOS by the other (ADC/NSCLC).
After review of data from the ancillary panel, all cases
with initially agreed diagnoses of ADC and SQCC remained

unchanged. Of the 14 cases classified initially as NSCLCNOS/NSCLC-NOS, four were reclassified as ADC by both
pathologists (ADC/ADC) (Figure 1), three as SQCC by both
pathologists (SQCC/SQCC) (Figure 2) and four to ADC by
one pathologist (NSCLC/ADC). Three cases remained as
NSCLC-NOS/NSCLC-NOS, all three samples being cell pellets derived from TBNAs. In the seven initially discordant
cases, all four SQCC/NSCLC were reclassified by both pathologists as SQCC, and two of the three NSCLC/ADC cases
by both pathologists as ADC (Figure 3).
Assessment of all data from all three pathologists showed
no cases where there was a discordant diagnosis of ADC and
SQCC in the same patient after ancillary investigation.
Ten cases with a diagnosis of ADC were subsequently
sent for gene mutation analysis. In all of these specimens,
sufficient DNA was present to be able to detect activating
EGFR mutations down to a sensitivity of at least 10%
(ranging from 1 to 10%). Sensitivity was calculated based on
the quantity and quality of the DNA used for EGFR mutation
analysis. None showed EGFR mutations (Table 1).

DISCUSSION
Most patients with NSCLC present with advanced
disease and require systemic therapy. For these, the di-

FIGURE 1. (A) A case of non-small cell lung carcinoma on TBNA of lymph node shows (B) undifferentiated tumor cells in the
cell pellet that stain for (C) TTF-1, with reclassification as adenocarcinoma.

FIGURE 2. A case of non-small cell lung carcinoma on endobroncial biopsy is reclassified as squamous cell carcinoma
after immunohistchemistry. (A) H&E, (B) P63 positive (left side of field), and (C ) CK5/6 positive (left side of field). Note
the focal positive native epithelial cells in (B) and (C ) (right side of field) that need careful interpretation to avoid false
positive diagnoses.

438

Copyright 2010 by the International Association for the Study of Lung Cancer

Journal of Thoracic Oncology Volume 5, Number 4, April 2010

Refining the Diagnosis and EGFR Status of NSCLC

FIGURE 3. Validation by two pathologists shows greater concordance of diagnoses for either SQCC or ADC from 11 to 24
out of 32 cases, through usage of an ancillary panel comprising thyroid transcription factor-1 (TTF-1), cytokeratin 5/6 (CK5/6),
P63, and a diastase-resistant periodic acid Schiff (D-PAS) stain for mucin. Yellow boxes are diagnoses on routine staining at
first review. Green boxes are diagnoses after review of ancillary panel. Adenocarcinoma (ADC)/ADC, squamous cell carcinoma
(SQCC)/SQCC, non-small cell carcinoma-not otherwise specified (NSCLC-NOS)/NSCLC-NOS denotes agreed diagnoses by validating pathologists of ADC, SQCC and NSCLC-NOS, respectively. SQCC/NSCLC-NOS denotes classification as SQCC by one
pathologist and NSCLC-NOS by the other. ADC/NSCLC-NOS denotes classification as ADC by one pathologist and NSCLCNOS by the other.
TABLE 1. EGFR Mutation Analysis on Small Biopsies
ID

Sample Type

EGFR Status

Sensitivity (up to), %

1
2
3
4
5
6
7
8
9
10

TBNA pellet
TBNA pellet
TBNA pellet
TBNA pellet
Endobronchial biopsies
TBNA pellet
Endobronchial biopsies
Pleural eff pellet
TBNA pellet
Pleural eff pellet

Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type

1
15
1
15
15
510
5
1
1
15

EGFR, epidermal growth factor receptor; TBNA, transbronchial needle aspirate;

Eff, effusion.

chotomization of NSCLC subtype into squamous and

nonsquamous has become important in recent years, with
the advent of pemetrexed and bevacizumab therapy. This
trend is continuing with other agents being developed with

differential efficacy by NSCLC subtype, for example insulin-growth factor receptor type 1 inhibitors show great
promise in squamous subtype tumors.17
This study shows that a small immunohistochemistry
panel of TTF-1, CK 5/6, and P63, together with a mucin stain,
refined diagnosis of NSCLC-NOS to either ADC or SQCC in
65% of cases in the test phase. This included 6 of 13 samples
taken via TBNA, with subsequent cell pellet formation from the
remaining fluid. This ancillary panel also produced greater
agreement and refinement of diagnosis on independent validation by two further pathologists at ends of the spectrum of
expertise in pulmonary pathology. Of note, immunohistochemistry produced no cases with conflicting data where ADC and
SQCC were diagnosed in the same patient.
Although several antibodies are described as being
markers of squamous differentiation (P63, CK5/6, and
34E12) and adenocarcinomatous differentiation (TTF-1),
there is very little published literature in relation to its usage
in small biopsies in the lung, even more so with cytology
preparations, specifically TBNAs. Studies have shown that
TTF-1 and P63 are useful in distinguishing between SCLC

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Journal of Thoracic Oncology Volume 5, Number 4, April 2010

Nicholson et al.

and poorly differentiated SQCCs,9,10 although other have

reported P63 staining in one third of ADCs on a study using
tissue microarrays.18 Our data suggest that the both these
antibodies are consistently of value in distinguishing ADC
from SQC. One recent study in relation to a trial of bevacizimab reported that that P63 and CK5/6 were useful in
distinguishing SQCC from ADC in fine needle aspirate specimens,19 data to support our findings. Our findings also
highlight the value of staining for TTF-1 and the technically
more simple investigation of looking for mucin in identifying
ADC. Evidence of mucin production has long been recognized as a good marker of adenocarcinomatous differentiation16,20 but has often been forgotten in recent years with
advances in immunohistochemistry. With positive staining in
approximately 60% in pulmonary ADCs, this should not be
forgotten as a useful test.
The prime criticism of this study is that resections are
not available for confirmation of classification. However, all
of these antibodies are well described as being either markers
of squamous differentiation8,10,11 or adenocarcinomatous differentiation14,15 in resection specimens. Furthermore, one
recent study of resection specimens diagnosed as ADC, SQC
or large cell carcinoma, for whom an initial biopsy was
diagnosed as NSCLC only, showed high specificities and
sensitivities for the four markers in our panel in relation to
correct assignment of diagnosis on biopsy to SQC or ADC.21
It is therefore reasonable to conclude that our data on biopsies
and also on tumor cells from TBNAs are a true reflection of
tumor type, given that the current pathologic classification for
lung neoplasms solely relates to resections,16 whereas 85% of
patients only have biopsy material. Indeed an International
Association for the Study of Lung Cancer/American Thoracic
Society/European Respiratory Society-sponsored multidisciplinary workshop on ADCs is ongoing with one of its remits
being to address this issue. Our data also demonstrate that
staining for high molecular weight CKs (34E12) showed a
much greater frequency and extent of staining across the
spectrum of NSCLCs with many of the ADC samples being
positive, suggesting limited use for this antibody in this
context. Others have however suggested that this antibody
may be useful in this context.22
A second issue is tumor heterogeneity in that, as in this
study, SQCCs may contain very occasional mucin vacuoles
and no antibody is 100% specific. In addition, more central
ADCs, which encompass the nonterminal respiratory unit
type ADCs,23 will be negative for TTF-1, however still
positive with a mucin stain in some cases. On this basis,
although there is similarity in staining profiles between
CK5/6 and P63 and overlap between TTF-1 positivity and the
presence of mucin within tumor cells in ADCs, we currently
favor the usage of all these tests. Further studies may refine
our practice in due course.
We extended analysis of patients with a final diagnosis
of ADC to assess whether biopsy material, especially from
cytologic/TBNA samples, are sufficient for somatic genotyping. All 10 specimens analyzed contained sufficient DNA of
adequate quality and concentration, but no EGFR mutations
were detected. With the incidence of EGFR mutations rang-

440

ing from approximately 10% in populations outside the far

East24,25 this is likely to simply reflect a true absence given
the small numbers analyzed, because the genotyping methodology used has a dilutional sensitivity lower limit of
approximately 1%. However, our numbers are small and
continued investigation of larger cohorts is therefore required
before such samples can be viewed as routinely adequate for
therapeutic decision making.
Finally, although immunohistochemistry undoubtedly
refined a diagnosis of NSCLC, there remained cases, especially those derived from TBNA, where the final diagnosis
remained as NSCLC-NOS due primarily to a lack of immunostaining precluding more accurate diagnosis, due to either
small volumes of tumor or heavy contamination by native
epithelial cells in TBNAs. In addition, there were three cases
with marked pleomorphism that highlight issues in relation to
extrapolation of a classification system based on resections to
small biopsies. One could argue that such cases should be left
as NSCLC on morphology due to the likelihood of them
being pleomorphic carcinomas, whereas their immunohistochemical profiles led to all three being preferably classified as
ADC, perhaps a better refinement of diagnosis in terms of
potential systemic therapy options. This highlights the importance of future classifications having input from disciplines
other than pathologists so that lung cancer classification
remains relevant to all user groups, and future reviews of the
WHO classification will need to address such issues as and
when they arise.

ACKNOWLEDGMENTS
SP is in receipt of a Clinical Senior Lectureship Award
from the Higher Education Funding Council for England. SP
and DCG also acknowledge NHS funding to the Royal Marsden
Hospital/Institute of Cancer Research NIHR Biomedical Research Centre.
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