2998 Vol. 9, 2998 –3003, August 1, 2003
Clinical Cancer Research
LKB1 Protein Expression in the Evolution of Glandular Neoplasia
of the Lung
Hassan Ghaffar, Fikret Sahin,
Montserrat Sanchez-Cepedes, Gloria H. Su,
Marianna Zahurak, David Sidransky, and
William H. Westra1
Departments of Pathology [H. G., F. S., G. H. S., W. H. W.],
Oncology [G. H. S.], OncologyBiostatistics [M. Z.], and
Otolaryngology-Head and Neck Surgery [D. S., W. H. W.], The Johns
Hopkins Medical Institutions, Baltimore, Maryland 21231, and the
Molecular Pathology Program, Spanish National Cancer Center,
Madrid, Spain [M. S-C.]
ABSTRACT
Purpose: About one-third of sporadic lung adenocarcinomas demonstrates biallelic inactivation of the LKB1 gene,
but the timing of this event is not known.
Design: We performed LKB1 immunohistochemistry
on 35 primary lung adenocarcinomas and 96 atypical adenomatous hyperplasias (AAH), a form of early glandular
neoplasia from which some lung adenocarcinomas arise.
Results: In all cases, strong cytoplasmic staining was
noted in the non-neoplastic epithelium lining the airways
from the bronchi to the terminal bronchioles. There was a
marked reduction in LKB1 staining in 9 of 35 (26%) adenocarcinomas and in 10 of 96 (10%) AAHs. When the AAHs
were subclassified on the basis of cytoarchitectural atypia,
loss of LKB1 expression was more frequent in the highgrade lesions (7 of 33, 21%) than low-grade lesions (3 of 63,
5%; P ⴝ 0.021). For the 21 adenocarcinomas where the
genetic status was known, immunohistochemistry staining
reliably reflected the activational state of the LKB1 gene
(95% concordancy).
Conclusions: In AAH, loss of LKB1 expression is
strongly associated with severe dysplasia, suggesting that
LKB1 inactivation may play a role in the critical transition
from premalignant to malignant tumor growth.
INTRODUCTION
Lung cancer remains the leading cause of cancer-related
mortality in the United States for both men and women. Approximately 172,000 new cases of lung cancer will be diagnosed
this year. Of those affected, 85% will not survive 5 years (1).
Received 11/5/02; revised 1/24/03; accepted 2/3/03.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1
To whom requests for reprints should be addressed, at The Weinberg
Cancer Center, Room 2242, 401 North Broadway, Baltimore, MD
21231. Phone: (410) 955-2163; Fax: (410) 955-0115; E-mail: wwestra@
jhmi.edu.
Adenocarcinomas now represents the most frequent subtype of
lung cancer (2), and they are usually discovered late in the
course of the disease even in the setting of vigilant radiographic
and cytologic screening (3). Novel strategies based on the detection of genetic markers offer new hope for improved risk
assessment, early cancer detection, and tumor surveillance, but
the impact of these strategies has been limited by an incomplete
understanding of the biology of lung cancer. Disappointedly,
only a few genetic alterations critical to the development of lung
adenocarcinomas are currently recognized, and the timing
of these alterations during glandular neoplasia remains to be
delineated.
LKB1, also known as STK11, is a serine/threonine kinase that functions as a suppressor of tumor growth. Germline mutations of LKB1 cause Peutz-Jeghers syndrome, an
autosomal-dominant disorder characterized by gastrointestinal hamartomatous polyposis, mucocutaneous pigmentation,
and an increased risk of intestinal malignancies (4 – 6). Importantly, patients with germ-line mutations are also at an
increased risk of developing extra-intestinal malignancies,
including occasional lung adenocarcinomas (7–9). As a tumor suppressor gene, LKB1 may also be a target of inactivation in the evolution of certain sporadic malignancies.
Indeed, we recently reported mutations in about one-third of
sporadic lung adenocarcinomas, implicating LKB1 inactivation as one of the more frequent and important genetic events
in the development of lung adenocarcinomas (10).
AAH2 is a precursor lesion from which some lung adenocarcinomas arise (11, 12). They represent localized proliferations of enlarged and atypical epithelial cells lining intact alveolar septae and are usually noted as incidental microscopic
findings in lungs resected for primary adenocarcinomas. Morphological and genetic evidence suggests that AAH encompasses a heterogeneous population of lesions representing
different points along the progression toward overt lung adenocarcinoma (13, 14). Under the light microscope, the cytological
and architectural atypia that characterize AAH falls along a
spectrum ranging from mild to severe (13–15). At the genetic
level, the frequency of p53 alterations and loss of heterozygosity
at 3p, 9p, and 17p tend to increase in proportion to the severity
of atypia (13–17). As such, AAH is now valued as a good target
for studying the timing and sequence of the genetic alterations
driving multistep lung tumorigenesis, and it represents a useful
lesion for addressing the timing of LKB1 inactivation in the
development of lung adenocarcinomas.
2
The abbreviations used are: AAH, atypical adenomatous hyperplasia;
IHC, immunohistochemistry; BAC, bronchioloalveolar carcinoma.
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Clinical Cancer Research 2999
Fig. 1 Histological progression and LKB1 staining of AAH. Low-grade AAH characterized by small atypical cells lining a delicate intersitium (A).
In high-grade AAH, there is cell crowding, increased cytologic atypia, and more prominent thickening of the septal walls (C). LKB1 staining is limited
to the atypical cells and not seen in the adjacent normal lung parenchyma in both the low-grade (B) and high-grade (D) lesions.
MATERIALS AND METHODS
Patients and Lesion Classification. One component of
the study group consisted of 15 patients identified through a
review of the histopathologic slides of all lung resections performed at The Johns Hopkins Hospital from 1995 to 2002. From
the lung resections of these 15 patients, 96 AAHs and 14 paired
primary lung cancers were available for LKB1 IHC. The other
component of the study group consisted of 21 patients with
primary lung adenocarcinomas that were already well characterized with respect to LKB1 gene as reported previously (10).
Briefly, 30 primary lung carcinomas had been screened using an
extensive panel of highly polymorphic microsatellite markers
that map to the LKB1 region of chromosome 19p. Of these, 21
carcinomas (all adenocarcinomas) showed loss of heterozygosity at 19p and on this basis were selected for complete analysis
of the LKB1 gene to search for: (a) point mutations in all exons
and intron– exon boundaries; (b) partial and complete deletions
and insertions; and (c) gene promoter hypermethylation using
methylation sensitive PCR. The medical records and histopathologic slides were reviewed to obtain information regarding
stage, grade, and type of the lung carcinomas.
The AAHs were identified using the criteria of Nakanishi
(18). Specifically, AAHs were identified as a growth of cytologically atypical cuboidal to columnar cells along the alveolar
septa. Lesions were not included that showed frank invasion of
the lung parenchyma or if they showed direct contiguous extension from a primary lung carcinoma. The AAHs were graded by
one of us (W. H. W.) without knowledge of LKB1 staining,
using criteria established previously (13–15). AAHs classified
as low grade were characterized by a single layer of round to
cuboidal cells with low to moderate cellular density; small cell
size; minimal variation in cell size, shape, and chromaticity; and
minimal thickening of the alveolar septa (Fig. 1A). By comparison, AAHs classified as high grade had increased cellular
density, larger cell size, greater variation in cell size and shape,
and thickening of the alveolar septa (Fig. 1C). Although some of
these high-grade lesions showed striking cytoarchitectural atypia, they were very small and did not show invasion of the lung
parenchyma.
LKB1 IHC. Sections (5 ␮m) were deparaffinized,
treated with sodium citrate epitope retrieval buffer (Ventana-Bio
Tek Solutions; Ventana Medical Systems, Tucson, AZ), and
then steamed for 20 min at 90°C. After cooling for 5 min, the
slides were labeled with a rabbit polyclonal anti-LKB1 antibody
(Cell Signaling Technology, Beverly, MA) using the Bio TekMate 1000 automated stainer (Ventana Medical Systems). This
anti-LKB1 antibody recognizes the COOH terminus of human
LKB1. Labeling with the primary antibody was carried out at a
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3000 LKB1 Immunohistochemical Staining in Glandular Neoplasia
Fig. 2 LKB1 immunohistochemical staining in nonneoplastic lung. The epithelium
lining the airways exhibits
strong cytoplasmic staining.
1:20 dilution. The LKB1 antibody was visualized using the
avidin-biotin technique (DAKO LSAB Kit; DAKO Cytomation,
Carpinteria, CA).
The non-neoplastic airway epithelium served as an internal
positive control. For the AAHs and adenocarcinomas, the following criteria were used to guide measurement of LKB1 staining: (a) interpretation was limited to areas of the neoplasm
adjacent to normal airways, such that staining intensity could be
interpreted relative to internal control; (b) LKB1 staining was
classified as positive if the staining intensity in the neoplasms
matched or exceeded the staining intensity of the normal airway
epithelium; and (c) conversely, LKB1 staining was classified as
negative if the staining intensity in the neoplasms was reduced
relative to the normal airway epithelium. A pancreatic adenocarcinoma with a known homozygous deletion of chromosome
19p encompassing the LKB1 gene served as a negative control.
Staining was interpreted as positive or negative without knowledge of the genetic status of the LKB1 gene.
RESULTS
We tested 96 AAHs and 35 primary lung adenocarcinomas
for LKB1 inactivation by IHC. The 96 AAHs included 63
low-grade and 33 high-grade lesions. An example of each AAH
category is shown in Fig. 1. The AAHs ranged in size from 0.1
to 7 mm. The mean size of the low-grade lesions was 1.5 mm.
The mean size of the high-grade lesions was 3.3 mm. All of the
AAHs were incidental histological findings in lung resection
specimens from 15 patients. One of these patients had a lung
resection for non-neoplastic disease, and the other 14 had lung
resections for primary lung carcinomas. All of the lung carcinomas were adenocarcinomas, including 2 nonmucinous BACs
and 12 non-BACs. Of the 21 lung adenocarcinomas that were
not associated with AAH, 4 were BACs, and the other 17 were
non-BACs. The pathologic stage of the lung carcinomas ranged
from T1 (n ⫽ 28) to T2 (n ⫽ 5) to T3 (n ⫽ 1) to T4 (n ⫽ 1),
and the histological grade ranged from well (n ⫽ 13) to moderate (n ⫽ 16) to poor (n ⫽ 6).
In the non-neoplastic lung tissue, strong LKB1 staining
was limited to the epithelium lining the airways from the large
central bronchi to the terminal bronchioles (Fig. 2). Staining
tended to be diffuse throughout the cytoplasm, but there was
occasional accentuation of staining in the apical cytoplasm of
the ciliated columnar cells. The only other cell type that demonstrated strong LKBI staining was the chromogranin-positive
neuroendocrine cell (i.e., Kulchitsky cells). LKB1 staining was
observed in the normal epithelium lining the pancreatic ducts,
but it was not observed in the control pancreatic adenocarcinoma that harbored a homozygous deletion of chromosome 19p
encompassing the LKB1 gene.
LKB1 staining of the epithelium lining the alveolar septa
was weak or entirely absent. In striking contrast, 86 of the 96
(90%) AAHs demonstrated strong LKB1 staining (Fig. 1, B and
D). Although negative LKB1 staining was not common in AAH
overall, negative staining was much more frequent in high-grade
lesions. Loss of LKB1 expression was noted in only 3 of the 63
(5%) low-grade lesions but in 7 of 33 (21%) high-grade lesions.
In the overly malignant lung adenocarcinomas, negative LKB1
staining was noted in 9 of 35 (26%) tumors (Fig. 3). LKB1
protein inactivation increased in frequency with progression
from low-grade AAH to high-grade AAH to overt adenocarcinoma (Fig. 4). The differences in LKB1 staining in this progression were significant (P ⫽ 0.005, Cochran-Armitage test for
trend). By logistic regression estimates, differences in staining
were significant when the low-grade AAH was compared with
high-grade AAH (P ⫽ 0.021) and adenocarcinoma (P ⫽ 0.006).
The differences in LKB1 staining were not significant between
high-grade AAH and lung adenocarcinoma (P ⫽ 0.662). There
was no relationship between LKB1 staining in an AAH and its
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Clinical Cancer Research 3001
Fig. 3 LKB1 staining in lung adenocarcinoma.
A, this tumor (ⴱ) shows cytoplasmic staining of the
intensity seen in the adjacent normal airway epithelium (arrow). B, this tumor (ⴱ) shows a marked
reduction in staining compared with the adjacent
normal airway epithelium (arrow).
paired lung carcinoma. In other words, LKB1 expression in the
primary lung carcinoma was not predictive of LKB1 staining in
the paired AAHs. For the lung adenocarcinoma, there was no
correlation between LKB1 staining and tumor subtype (BAC
versus non-BAC), grade, or stage.
For the 21 adenocarcinomas where the genetic status was
known, LKB1 IHC staining reliably reflected the activational
state of the LKB1 gene (Table 1). Fifteen of the lung adenocarcinomas did not show biallelic inactivation, and all of these
tumors exhibited LKB1 staining. Conversely, negative LKB1
staining was noted in 5 of the 6 of the lung adenocarcinomas
that demonstrated biallelic gene inactivation.
DISCUSSION
Lung cancer doggedly persists as the leading cause of
cancer-related mortality (1). Novel strategies based on the molecular genetic profiling of lung cancer offer renewed hope for
early cancer detection and effective therapy, but recognition of
the genes responsible for lung tumorigenesis remains a daunting
challenge. LKB1 is a tumor suppressor gene that encodes a
serine/threonine kinase (4 – 6). Germ-line mutations of LKB1
typically give rise to oral mucocutaneous hyperpigmentation,
intestinal hamartomas, and gastrointestinal malignancies, but
there is also an increased risk of extraintestinal malignancies,
including occasional lung adenocarcinomas (7–9). Inactivation
of LKB1 is also observed in some sporadic lung adenocarcinomas (19). We have shown previously that about one-third of
lung adenocarcinomas harbor inactivating alterations of the
LKB1 gene, resulting in complete loss of protein expression or
the production of a truncated protein (10). Thus, alterations of
the LKB1 tumor suppressor gene appear to be one of the more
common and relevant genetic events in the development of lung
adenocarcinoma. In the present study, we used an immunohistochemical approach to determine the distribution of LKB1
protein expression in the lung and various stages of glandular
neoplasia of the lung.
The expression of LKB1 in human adult tissues remains to
be more fully characterized. Previous studies using Northern
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3002 LKB1 Immunohistochemical Staining in Glandular Neoplasia
Table 1
Correlation between biallelic inactivation of the LKB1 gene
and LKB1 protein expression as determined by
immunohistochemistrya
LKBI
Fig. 4 Loss of LKB1 expression across various stages of glandular
neoplasia of the lung. Loss of LKB1 expression increases in frequency
from low-grade AAH to adenocarcinoma. The difference in frequency
of LKB1 expression was statistically significant (P ⫽ 0.005).
blot and reverse transcription-PCR analyses indicate that LKB1
is widely expressed in most adult tissue types (5, 6), but the
cell-specific patterns of LKB1 expression are difficult to discern
from studies of gross tissue extracts. In situ and immunohistochemical methods for LKB1 expression permit greater resolution of LKB1 localization within adult tissues. Using an in situ
hybridization approach, Rowan et al. (20) confirmed widespread expression in adult tissues, including the lung. In addition, they observed that LKB1 mRNA expression was: (a)
strictly confined to the epithelia; (b) increased in cells undergoing continuous or rapid division; and (c) lost in a subset of
carcinomas. We were able to validate these findings using an
immunohistochemical approach. In the non-neoplastic adult
lung, LKB1 protein expression was confined to the epithelium
lining the airways. Cytoplasmic staining tended to be strong and
intense from the large bronchi to the terminal bronchioles.
LKB1 protein expression was considerably attenuated in the
more static epithelial cells lining the alveoli (i.e., pneumocytes
and Clara cells).
Just as the immunohistochemical observations support
some widespread functional role for LKB1 protein expression in
epithelial tissues of the lung, they also suggest that loss of this
functional activity may play some role in the pathogenesis of a
subset of sporadic lung adenocarcinomas. A portion (26%) of
the lung adenocarcinomas demonstrated absent or reduced
LKB1 protein expression by IHC, a frequency predicted by the
known prevalence of biallelic gene inactivation in lung adenocarcinomas (10). Indeed, there was strong correlation between
status of the LKB1 gene and predicted immunohistochemical
staining. Of the 15 lung adenocarcinomas that did not show
biallelic inactivation of the LKB1 gene, all demonstrated strong
LKB1 staining. Conversely, of the 6 lung adenocarcinomas with
biallelic inactivation of the LKB1 gene, 5 showed negative
LKB1 staining. Disparity between LKB1 gene status and protein
expression was noted in only a single case (case 4), where strong
staining was noted in a carcinoma that demonstrated loss of
heterozygosity in the region of LKB1 and a point mutation
giving rise to a stop codon.
Case
Biallelic gene inactivation
Protein expression
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Negative
Negative
Negative
Positive
Negative
Negative
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
a
Biallelic inactivation as evidenced by loss of heterozygosity for
microsatellite markers near the LKB1 locus, coupled with an LKB1
non-sense mutation or promoter hypermethylation as reported previously.
For some critical tumor suppressor genes, such as p53,
inherent sensitivity and specificity problems render IHC an
unreliable indicator of true gene status (21). This does not
appear to be the case for LKB1 IHC. Although LKB1 IHC may
underestimate the true incidence of biallelic inactivation in lung
adenocarcinomas (as a result of tumor heterogeneity, binding of
a polyclonal antibody to truncated protein, or some other mechanism), overall, it is a reliable method for assessing LKB1 gene
status. As IHC is rapid, inexpensive, and widely available to
most diagnostic laboratories, this method may circumvent the
need for more cumbersome genetic evaluation of the LKB1,
particularly when sufficient quantities of tumor-rich DNA are
difficult to obtain. As one example, IHC may be a suitable
method for addressing the role of LKB1 inactivation in the
earliest stages of glandular neoplasia, such as AAH, a lesion that
has been recalcitrant to exhaustive molecular genetic analysis
because of its small size.
As an early stage of glandular neoplasia, AAH is valued as
a good target for delineating the timing and sequence of genetic
alterations in the development of lung adenocarcinomas. In turn,
the study of AAHs has been helpful in formulating a molecular
genetic progression model for certain lung cancers (11). According to the current understanding of glandular neoplasia of
the lung, activation of the K-ras oncogene seems to be an early
event involved in the initiation of AAH (12, 22). Progression of
AAH through increasing degrees of morphological dysplasia
requires the silencing of key tumor suppressor genes, such as
p16 (17). Ultimately, activation of telomerase (23) and inactivation of the p53 tumor suppressor gene (13–16) appear to be
important in triggering invasive tumor growth. Like p53, inac-
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Clinical Cancer Research 3003
tivation of LKB1 likely participates in the critical transition from
premalignant to malignant tumor growth. LKB1 expression is
generally very high in AAH relative to its expression in normal
epithelial cells lining the alveoli. Negative LKB1 staining was
detected in only a small percentage of AAHs. However, when
AAH is subclassified on the basis of its morphological features,
negative staining increases with the severity of cytoarchitectural
atypia. Negative staining was observed in only 5% of the lowgrade lesions but in ⱕ21% of the high-grade lesions.
The natural history of early glandular neoplasia is not well
understood. In particular, the frequency and pace at which AAH
progresses to overtly malignant lung adenocarcinoma are not
known. Longitudinal observation of AAH now permitted by
high-resolution imaging techniques indicates that the mere presence of AAH does not necessarily indicate sure and unremitting
progression to adenocarcinoma (24). Indeed, AAHs appear to
represent a morphologically and genetically diverse group of
lesions with varying potentials for malignant transformation (13,
25). Unraveling the molecular genetic alterations that initiate the
development and drive the progression of these lesions might
provide new insight into the biology of lung cancer and facilitate
the design of novel strategies for early detection of lung cancer.
Alterations of LKB1 function may represent one of the critical
steps in the transition from a benign to a potentially malignant
proliferation of pneumocytes.
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LKB1 Protein Expression in the Evolution of Glandular
Neoplasia of the Lung
Hassan Ghaffar, Fikret Sahin, Montserrat Sanchez-Cepedes, et al.
Clin Cancer Res 2003;9:2998-3003.
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