Cancer
Letters101 (1996) 211-217
Alveolar stem cells in canine bronchial carcinogenesis
A.A.W. Ten Have-Opbroeka,b,*,J.R. Benfieldb, W.G. Hamrnondb,J.H. Dijkmana
aDepartments of Pnevnwlogy, L.&den University, Leiden, The Netherlands
bCardiothoracic Surgery, University of California, Davis, USA
Received12December1995;accepted3 January1996
Abstract
Alveolar type II cells are not presentin normal epithelium of canine segmentalbronchi but after carcinogen exposure they
do occur in intra-epithelial lesions with all degreesof atypia and in invasive lesions with different glandular growth patterns.
Immunohistochemistry for proliferation markers(F’CNA; Ki-67) strongly suggestthat such novel type II cells are pluripotential stem cells in canine bronchial carcinogenesis.Very likely, bronchial carcinogen&s is subject to an oncofetal mechanism
of differentiation: bronchial epithelial retrodifferentiation followed by novel differentiation of alveolar tumor stemcells.
Keywords: Bronchial carcinogenesis;Stem (progenitor) cells; Alveolar type II cells; Proliferating cell nuclear antigen; Nonsmall cell lung cancer; Breast/prostatecarcinoma
1. IntrodIlction
Information about stem cells for bronchogenic
carcinoma has remained inconclusive. Possible candidates are bronchial epithelial cells that are capable
of division [ 1,2]. previous studies in our canine
model of bronchial carcinogenesis (see below) suggest that the alveolar type II cell may be another
candidate [3-51. Such cells occur in major subsets of
bronchogenic carcinoma in humans [6-91 and animals (reviewed in [4]). Type II cells are actively proliferating phnipotential stem cells in normal mam* Comsponding anthor. RespiratoryBiology Group, Departmentof Anatomyand Embryology,L&en University,P.O.Box
9602, 2300 RC Leiden. The Netherlands. Tel.: +31 71
5276692/5276660/526295~
fax: +31 71 5276511; e-mail:
have@ruIK?.leidenuniv.nl
malian lung morphogenesis [ lO-121. They display
highly distinguishing features, including an approximately cuboid shape, a large and round&h nucleus,
cytoplasmic staining for SP-A, and presence of precursory or mature forms of multiiameI1a.r bodies [ lO141. In this report we demonstrate that type II cells
may play a role as pluripotential stem cells in bronchial carcinogenesis. However, as wiil be discussed,
further evidence is required to prove this conclusion
incontrovertibly.
2. Definitions
As defined [3-6], the term ‘cell of origin’ refers to
the very first undifferentiated (primor&&like)
tumor
progenitor cell that appears during transformation
(via metaplasia) of normal bronchial epithefium to
bronchogenic Icarcinoma. We use the term ‘stem cell’
0304-3835/%/$12.00Q 1996 ElsevierScienceIrelandLtd. All rightsreserved
PII: SO304-3835(96)04137-7
212
A.A. W. Ten Have-Opbroek et al. /Cancer Letters IO1 (1996) 211-217
to indicate the predominant proliferating cell type
that occupies the dividing layers of the (pre)neoplastic lesions.
3. Materials and methods
Canine subcutaneousbronchial autografts (SBAs)
are obtained from the bronchial tree of the left lung
following pneumonectomy; the major bronchi are cut
into segments and placed subcutaneously onto the
back through two short midline incisions (10-14
SBAs per dog). Approximately 6-8 weeks later, 3methylcholanthrene in sustainedreleaseimplants was
placed in the SBAs. As reported [15,16], carcinomas
developed in 77% (86/111) of these autografts; metastasesoccurred in two dogs. Tumors were successfully serially transplanted in athymic nude mice. The
bronchial mucosa of each SBA in which there was
ongoing carcinogenesis was sampled repeatedly and
serially. The intra-epithelial and invasive lesions described here were excised from the SBAs after 6-12
and 16-24 months exposure to carcinogen, respectively.
After fixation in formalin and embedding in paraffin, serial sections were cut at 6pm and placed on
poly-L-lysine coated slides. Adjacent sections were
used for immunohistochemistry and hematoxylin and
eosin (H&E) staining, which allowed for morphologic characterization of immunoreactive cells and
histological typing of the lesions [ 17,181.For immunohistochemistry, we used antibodies to: (1) proliferating cell nuclear antigen (PCNA), clone PC10
(Signet Laboratories, Dedham, MA) and Ki-67 proliferating cell antigen, clone MIB-1 (AMAC Inc.,
Westbrook, ME), mouse anti-human; and (2) natural
and recombinant human SP-A, rabbit or mouse antihuman (our antibody SALS-Hu [19,20]); gifts from
BYK Gulden Pharmaceuticals, Konstanz, Germany
[21] and other investigators). SALS-Hu was prepared
in rabbits as reported [19]; the SP-A specificity was
assessedby immunohistochemistry, Western blotting,
and in vitro translation of mRNA from human lungs
followed by immunoprecipitation [ 19,201.SALS-Hu
cross-reacts with SP-A in normal dog lung and canine adenocarcinoma as has been demonstrated by
immunohistochemistry [3-51 and Western blotting
[3]. The PCNA and Ki-67 antibodies were applied to
tissue sections according to the ABC-peroxidase
method using biotinylated horse anti-mouse secondary antibodies (Vector Laboratories, Burlingame,
CA), streptavidin peroxidase (Dako Corp., Carpinteria, CA), and 3,3-diaminobenzidine as the chromogen
[6,13]. Prior to incubation, endogenous peroxidase
activity was quenched with a freshly prepared 3%
hydrogen peroxide solution in methanol. Sections of
each specimen were processed for antigen retrieval
by microwave exposure in 10 mM citrate buffer (pH
6.0) [22]; for PCNA detection, adjacent untreated
sections were used as well. The optimal antibody
dilution in phosphate-buffered saline (PBS; pH 7.6)
(Sigma Chemical, St. Louis, MO) was assessedusing
positive normal tissue controls (colon; lymph nodes).
The Ki-67 antibodies were used at a 150 dilution,
and the PCNA antibodies at a dilution of 1:1000 for
microwave treated sections and of 1:200 for untreated sections.The antibodies to SP-A were applied
according to the indirect immunofluorescence technique [3-51 and the avidin/biotin complex (ABC)
method as above using swine anti-rabbit or horse
anti-mouse secondary antibodies (Vector Laboratories, Burlingame, CA) [6,13]. The sections were
counterstainedwith Mayer’s hematoxylin.
Immunohistochemical controls were performed on
the sametumor material with antibodies to Clara Cell
10 kDa protein (CClO) (rabbit anti-human; gift of Dr.
G. Singh, Pittsburg, PA), preimmunization serum
(PS) or normal mouse serum as the primary antibody
or omission of one of the incubation steps, and on
normal canine lung tissues.
4. Results
Lesions that had arisen from the existing normal
bronchial epitbelium of SBAs after carcinogen exposure showed abnormal epitbelial areas with mild to
severeatypia and in situ carcinoma, sometimesinterspersedwith (almost) normal epithelium, and microand macro-invasive adenocarcinomas. The intraepithelial lesion illustrated (Fig. 1A; H&E staining)
had two different areas:one area, which is designated
here by the term ‘dysplastic focus’ (arrowheads),had
basal hyperplasia and squamousmetaplasiawith mild
to moderateatypia, whereasthe other area had regular hyperplastic epithelium (on the right). Immunohistochemistry (Fig. 1B) revealed that both areas
contained PCNA-positive nuclei. This staining pat-
A.A. W. Ten Have-Opbroek et al. /Cancer Letters I01 (I 996) 21 l--2/ 7
713
Fig. I. (A) Intra-epithelial lesion from canine SBA showing a dysplastic focus (arrowheads) and regular hyperpiasnc epirhelium (on the
right). H&E x 170. (B) Nuclear staining for PCNA is present in the dysplastic focus and Ihe hyperplastic epithelium. ABC method, anti
PCNA x 155. (C) Detail of the dysplastic focus. The basal region contains cuboid cells with a large nucleus and little cytoplasm. IWE
x295. (D) Such cuboid cells are the major proliferating cells of the dysplastic focus. ABC method, anti PCNA X295. (E) The cuboid cells
in the dysplastic focus stain for SP-A, whereas the hyperplastic epithelium (on the right) does not stain. ABC method, anti SP-A X 190. (F)
Immunohistochemical controls are negative. ABC method, anti CC10 X 190. Formalin fixation; adjacent serial sections; hematoxylin
counterstaining.
tern was not influenced by the methods used (antigen
retrieval versus no antigen retrieval). A similar distribution pattern was found for Ki-67. Closer examination in the H&E-stained section (Fig. 1C) showed
that the dyspktic focus had two major categories of
epithelial cells. The approximately cuhoid cells,
which occupied the basal and adjacent layers, had a
large nucleus iand little cytoplasm. The nuclei were
A.A. W. Ten Have-Opbroek et al. /Cancer Letters IOI (1996) 211-217
Fig. 2. (A) Lesion of adenocarcinoma.The basal region contains cuboid cells with a large nucleus and little cytoplasm. H&E x240. (B), (C)
Such cuboid cells am the major proliferating cells of the lesion (B) and show cytoplasmic staining for SP-A (C). (B) ABC method, anti
PCNA x120. (C) Immunofluomscence,anti SP-A X275. (D) Immunohistochemical controls are negative. Anti CC10 x70. Formalin fixation; adjacent serial sections; hematoxylin counterstaining (A1B.D).
round to ovoid or sometimes irregular, lightly to
moderately or sometimes strongly basophilic, and
contained one or sometimes two prominent nucleoli.
The second category of cells, which occupied the
upper adluminal layers, often had more cytoplasm.
Their hyperchromatic nuclei were still rather large,
indicating that there was no true squamous maturation in the dysplastic focus. Nuclear PCNA (Fig. ID)
and Ki-67 staining predominated in the basal and one
or two adjacent epithelial layers of the dysplastic
focus, where mitotic figures were also seen. This
indicated that the cuboid cells with a large nucleus
and little cytoplasm, which occupied these layers (see
Fig. lC), were the major proliferating cells of the
dysplastic focus. As shown (Fig. lE), such cuboid
cells were SP-A positive, whereas the cells in the
regular hyperplastic area (on the right) did not stain.
The SP-A staining of the cuboid cells was delicate
and included the peripheral cytoplasm as also seen in
embryonic lungs [12], which suggests cellular immaturity in the lesions [3,4]. A weak SP-A staining was
sometimes found in the larger cells present in upper
layers of the dysplastic focus. Some staining found
along the luminal surface was probably caused by the
presence of SP-A which had been secreted into the
lesion’s lumen. However, as shown in immunohistochemical controls (see Fig. lF), it could also be nonspecific staining of necrotic material present on the
luminal surface. Immunohistochemistry for CC10
provided a positive staining of non-ciliated bronchiolar epithelial cells in normal canine lung but in preneoplastic lesions (Fig. lF), neither cuboid nor other
A.A. W. Ten Have-Opbroek et al. /Cancer Letters I01 (1996) 211-217
215
basophilic and roundish and often had one or sometimes two very prominent nucleoIi. Nuclear stirring
for PCNA (Fig. 2B) or Ki-67 usu&Iy predominated
in the basal and adjacent epithelial layers of the lesions. In a number of lesions, the nuokr stnining
was present in more upper layers as well. As appearedfrom the adjacent H&E-stirred sections, such
variant staining patterns were prob&Iy ca~4 by a
tangential cut of the lesions. The findings indicated
that the basally located cub&d cells wit& a large nucleus and little cytoplasm were the major proliferating cells of the lesions. As shown (Fig. ZC), all
cuboid cells of the basal epithelial layer and some
cuboid cells in upper layers stained for SP-A,
whereas the larger cells in the
region were
(almost) negative. Staining for CC10 (Fig. 2D) and
other immunohistochemical controls were completely
negative.
Fig. 3. Tumor type II cells often show immature multilameliar
bodies (shafted arrow), dense bodies (arrow) and osmiophilic
multivesicular bodies (arrowhead) as found in type II cells of
early fetal lungs. Glutaraldehyde/paraformaldehyde fixation.
Uranyl acetate/lead citrate x27 200. From Ref. [3], with permission of the American Society for Investigative Pathology.
epithelial cells displayed any staining. Other immunohistochemical controls were also negative.
A similar relationship between cell type (i.e.
cuboid cell with a large nucleus and little cytoplasm)
and specific protein (PCNAKi-67; SP-A) expressions as found in the dysplastic lesion above was
observed in other intra-epithelial lesions with mild to
severeatypia, in in-situ carcinoma, and in micro- and
macro-invasive lesions from adenocarcinomas with
different (bronchiole-alveolar; acinar; and other)
growth patterns.
Fig. 2 gives an example of our findings in adenocarcinomas. The cuboid cells, which occupied the
basal and adjacent epithelial layers of the lesions
(Fig. 2A; H&E staining), had a large nucleus and
little cytoplasm. The nuclei were round to ovoid or
sometimes irregular, moderately to strongly basophilic and contained one or sometimestwo nucleoli.
Occasionally, such cuboid cells were also found locally in more upper (adluminal) epithelial layers.
Larger cells with more abundant cytoplasm (Fig. 2A)
were present in upper epithelial layers as far as the
lumen, alone or in clusters, but never in basal epithelial layers. Their nuclei were lightly to moderately
5. Discussion
The data in this paper strongly suggestthat alveolar type II cells are pluripotential stem cells in canine
bronchial carcinogenesis(see Section 2). Such cells
are the predominant proliferating cells of Progressive
lesions which had arisen from canine SBAs after
carcinogen exposure, including intra-epithelid lesions with all degreesof atypia and invasive lesions
with different (bronchiole-alveolar; a&tar; and other)
glandular growth patterns. The type II stem cells
usually predominate in the basal region of the lesions
near the connective tissue but they may sometimes
occur in the upper region as well (present study) [35], which suggeststhat proliferation takes place in
both peripheral and luminal directions. However, as
shown, these cells may also differentiate b apparently viable larger cells with more abundant cytoplasm that is (almost) devoid of reactivity to SP-A.
Such larger cells occur exclusively in upper layers of
the lesions.
Light and electron microscopy and immunohistochemistry (present study) [3-51 demonstrate that alveolar type II cells involved in canine bronchial carcinogenesislack any similarity to other dividing cells
in the conducting airways such as mucous and basal
cells and the columnar secretory cell type in normal
distal bronchioles describedby Clara I23]. The tumor
type II cells have all the distinguishing features of
216
A.A. W. Ten Have-Opbroek et al. /Cancer Letters 101 (1996) 211-211
their normal counterparts, namely the approximately
cuboid cell shape, the large and roundish nucleus,
cytoplasmic staining for SP-A, and presence of precursory or mature forms of multilamellar bodies [lo141. Often, however, the cells look somewhat immature at the ultrastructural level [3] (see Fig. 3). In this
respect, they resemble type II cells present in early
fetal lungs [ 11,191 and in distal bronchioles of adult
lungs, the latter of which have been incorrectly considered to be Clara cells [ 12,131.
Type II tumor stem cells first appear in epithelium
of SBAs that is slightly abnormal (i.e. basal hyperplasia and squamous metaplasia with mild or moderate atypia) [3,4]. In the embryo, type II cells originate
from undifferentiated
primordial epithelium but
never from earmarked bronchial epithelium [lo].
Evidence that undifferentiated primordial cells exist
in the epithelium of larger airways after birth is not
available [lo]. In view of these data and present insights into embryonic lung differentiation [ 10-121,
we have postulated [4] that neoplastic progression for
adenocarcinoma development in SBAs may start with
local retrodifferentiation of existing normal bronchial
epithelial cells such as mucous or basal cells, which
results in the appearance of more or less undifferentiated (primordial-like) cells of origin. Differentiation
of type II tumor stem cells from such cells of origin
may occur by activation of genes that regulate type II
cell expression but were repressed in utero in earmarked bronchial cells, a phenomenon not unlike the
appearance of alpha fetoprotein in liver cancer. In
view of their way of induction (i.e. by carcinogen
exposure) and depending on other (growth, genetic,
environmental) factors, type II tumor stem cells
highly probably give rise to type II cell clones that
display significant variations in growth and differentiation potentials.
In normal mammalian lung morphogenesis, type
II cells have an enormous growth potential. They
generate the entire alveolar epithelial portion of the
lung and also maintain it in adulthood [lO-121. In
canine bronchial carcinogenesis type II cells give rise
to an active yet stable progeny with an apparently
high growth and invasive potential (present study)
[3-51. Very likely, the rates of proliferation, differentiation, and slough of type II tumor stem cells and
their descendants are responsible for the final size of
the lumen and the overall appearance of the adeno-
carcinomatous lesions. That such cells likely belong
to different clones (see above) may contribute to the
histologic complexity and diversity of adenocarcinomas. The present study does not exclude that also
unrelated clones, e.g. those produced by potential
tumor stem cells like mucous and basal cells, could
contribute to such pattern formation, although there
is no evidence for this possibility at this very moment. Our conclusion regarding a pluripotential stem
role of type II cells in canine bronchial carcinogenesis is in agreement with our findings of type II cell
populations in serial tumor transplants in nude mice
[3,5] and with our and other’s findings of type II
cells and SP-A protein or mRNA in bronchioloalveolar and other adenocarcinomas in humans and
animals [4,6-g]. Further characterization of the stem
cell clones involved in canine bronchial carcinogenesis is required to prove our conclusion incontrovertibly.
Our concept that the central duct system of the
lung may give rise to peripheral (alveolar) adenocarcinemas due to an oncofetal mechanism of differentiation (epithelial retrodifferentiation
followed by
novel differentiation) is novel and may be of interest
to carcinogenesis studies in other organs with a
prominent duct system, e.g. liver, pancreas, prostate,
mammary gland, and salivary glands. If our concept
is applicable, the duct system of, for instance, the
liver may play a more important role in carcinogenesis than has been expected from histologic typing of
carcinomas (mostly liver cell tumors). This may especially hold true for breast and prostate carcinomas,
in view of the impressive morphogenetic potential of
the duct systems in breast and prostate in postnatal
life (generation of the alveoli) which may enhance
disregulation. Further support for our concept may
come from studies of the regulation of epithelial expression in the duct system [24], especially in oncological animal models where lesions are induced at
selected focal sites [25].
Acknowledgements
The authors thank Mr. J.R. Johnson (Department
of Surgery, University of California Davis, USA) for
technical assistance, Dr. J.H.J.H. van Krieken
(Department of Pathology, Leiden University, The
Netherlands) for reading the manuscript, and Dr.
A.A. W. Ten Have-Opbroek et al. /Cancer Letters IO1 (19!Xi) 21 I-21 7
L.A. Russell (Department of Pathology, University of
California Davis, USA) for our discussions.
[13]
References
[l] McDowell, E.M. (1987) Bronchogenic carcinomas.In: Lung
Carcinomas, pp. 255-285. Editor: E.M. McDowell. Churchill Livingstone, Edinburgh.
[2] Nasiell, M., Auer, G. and Kato, H. (1987) Cytological studies in man and animals on development of bronchogenic
carcinoma. In: Lung Carcinomas,pp. 207-242. Editor: E.M.
McDowell. Churchill Livingstone, Edinburgh.
[3] Ten Have-Opbroek, A.A.W., Hammond, W.G., Bentield,
J.R., Teplitz, R.L. and Dijkman, J.H. (1993) Expression of
alveolar type II cell markers in acinar adenocarcinomasand
adenoid-cystic carcinomas arising from segmental bronchi.
A study in a heterotopic bronchogenic carcinoma model in
dogs. Am. I. Pathol., 142, 1251-1264.
[4] Ten Have-Opbroek, A.A.W., Benfield, J.R., Hammond,
W.G., Teplitz, R.L. and Dijkman, J.H. (1994) Invited review. In favour of an oncofoetal concept of bronchogenic
carcinoma development. Histol. Histopathol., 9, 375-384.
[S] Ten Have-Opbroek, A.A.W., Hammond, W.G. and Benfield,
J.R. (1990). Bronchiole-alveolar regions in adenocarcinoma
arising from canine segmental bronchus. Cancer Len, 55,
177-182.
[6] Ten Have-Opbroek, A.A.W., Dijkman, J.H., Van Krieken,
J.H.J.H., Boex, J.J.M., Hammond, W.G. and Benfield, J.R.
(1993) The presenceof alveolar type II cells in human bronchogenic carcinoma. Am. Rev. Resp. Dis., 147 (Suppl.),
A158.
[7] McDowell, E.M., McLaughlin, J.S., Merenyl, D.K., Kieffer,
R.F., Harris, C.C. and Trump, B.F. (1978) The respiratory
epithelium. V. Histogenesis of lung carcinomas in the human. J. Natl. Cancer Inst., 61,587-606.
[S] Linnoila, R.I., Mulshine, J.L., Steinberg, SM. and Gazdar,
A.F. (1992) Expression of surfactant-associatedprotein in
non-small-cell lung cancer: a discriminant between biologic
subsets.J. Natl. Cancer Inst. Monogr., 13.61-66.
[9] Tsutahara, S., Shijubo, N., Hirasawa, M., Honda, Y., Satoh,
M., Kuroki, Y. and Akino, T. (1993) Lung adenocarcinoma
with type II pneumocyte characteristics. Eur. Respir. J., 6,
135-l 37.
[IO] Ten Have-Opbroek, A.A.W. (1981) The development of the
lung in mammals: an analysis of concepts and findings. Am.
J. Anat.. 162, 201-219.
[Ill Ten Have-Opbroek, A.A.W. (1991) Lung development in
the mouseembryo. Exp. Lung Res., 17, 11l-130.
[12] Ten Have-Opbroek, A.A.W. and Plopper, C.G. (1992) Morphogenetic and functional activity of type II ceils in early
[14]
[IS]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
211
fetal Rhesusmonkey lungs. A comparison between primates
and rodents. Anat. Rec., 234,93-104.
Ten Have-Opbmek, A.A.W., Otto-Verbeme, C.J.M., Dubbeldam, J.A. and Dijkman, J.H. (1991) The proximal border
of the human respiratory unit, as shown by scanning and
transmission electron microscopy and light microscopical
cytochemistry. Anat. Rec., 229,339-354.
Plopper, C.G. and Ten Have-Gpbroek. A.A.W. (1994) Anatomical and Histological Classification of Bronchioles. In:
Diseasesof the Bronchioles, pp. 15-25, Editor: G.R. Epler.
Raven Press,New York.
Hammond, W.G., Bentield, J.R., Paladugu, R.R., Azumi, N.,
Pak, H.Y. and Teplitz, R.L. (1986) Carcinogenesisin heterotopic respiratory epithelium in canine subcutaneous bronchial autografts. Cancer Res., 46.2995-2999
Derrick, M.J., Hammond, W.G., Pak, H.Y., Azumi, N.,
Smith, S.S. and Benfield, J.R. (1988) Non-small cell lung
cancer in autogenoussubcutaneousbronchial grafts in dogs.
J. Thorac. Cardiovasc. Surg., 95,562-571.
Stiinzi, H., Head, K.W. and Nielsen, SW. (1974) lntemational histological classification of tumours of domestic
animals. I. Tumors of the lung. Bull. WHO, 50,9--19.
The World Health Organization (1982) HistologicaJ typing
of lung tumours,2nd edn. Am. J. Clin. Pathol.. 77. 123-136.
Otto-VerbernIe, C.J.M., Ten Have-Opbroek. A.A.W.,
Balkema, J.J. and Franken, C. (1988) Detection of the type
II cell or its precursor before week 20 of human gestation,
using antibodies against surfactant-associated proteins.
Anat. Embryol., 178,29-39.
Otto-Verbeme, C.J.M., Ten Have-Opbroek, A.A.W. and De
Vries, E.C.P. (1990) Expression of the major surfactantassociatedprotein, SP-A, in type II cells of human lung before 20 weeks of gestation. Eur. J. Cell Biol., 53, 23-19.
Voss, T., Eistetter, H., Schafer, K.P. and Engel, J. (1988)
Macromolecular organization of natural and recombinant
lung surfactant protein SP 28-36. J Mol. Biol.. 201, 219227.
Yu, C.C.W., Woods, A.L. and Levison, D.A. (1992) The
assessmentof cellular proliferation by immunohistochemistry: a review of currently available methods and their applications. Histochem. J., 24, 121-131.
Clara, M. (1937) Zur Histobiologie des Bronchalepithels. Z.
Mikrosk. Anat. Forsch.,41, 321-347.
Jetten, A.M. (1991) Growth and differentiation factors in
tracheobronchial epithelium. Am. J. Physiol.. 260 (Lung
Cell Mol. Physiol. 4). L361-L373.
Bentield, J.R. and Hammond, W.G. (1992) Bronchral and
pulmonary carcinogenesis at focal sites in dogs and hamsters.Cancer Res. (SuppI.), 52. 2687s-2693s