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Specialized Program of Research Excellence (SPORE) in Lung Cancer, University of Colorado Health Sciences Center, Denver, Colorado 80262 [R. M. G., H. A. D., T. C. K., S. P., W. A. F.], and Department of Pulmonary Sciences and Critical Care Medicine, Denver Veterans Affairs Medical Center, Denver, Colorado 80220 [R. L. K., Y. E. M., E. C. D.]
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ABSTRACT |
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 Top ABSTRACT IntroductionPatients and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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Strategies for early detection are changing as concepts of solid tumor carcinogenesis advance. The observation that many of the morphological and molecular abnormalities that exist in invasive tumors also occur to a lesser degree in preinvasive epithelium has led to the notion that many tumors, including lung carcinoma, are the result of a predictable progression of morphological and genetic changes in airway epithelium. This hypothesis has been especially well supported in the colon, where aberrant crypt foci have recently been recognized as the earliest of the precursor lesions that may eventuate in invasive carcinoma (6) . Histological changes associated with lung carcinogenesis include reserve cell hyperplasia, squamous metaplasia, low- or high-grade dysplasia (atypia), CIS3 , and invasive carcinoma. However, stepwise progression through this series of morphological changes is rarely observed in single individuals because premalignant airway lesions are less easily recognized and characterized than lesions in organs such as the colon.
The most accessible stage of bronchial carcinogenesis is invasive carcinoma. Genetic alterations are a nearly universal feature of invasive lung cancers and include LOH (typically of regions on chromosomes 3, 5, and 9) and point mutations in tumor suppressor genes (p53 and Rb) or oncogenes (K-ras). These genetic alterations have also been assessed in preinvasive bronchial epithelium from smokers. The most consistently observed abnormality in preinvasive epithelium from this group is allelic loss (LOH; Refs. 7, 8, 9, 10, 11 ). The high frequency of LOH in bronchial epithelium of smokers diminishes the potential utility of these specific alterations as predictive markers, and the overall prognostic value of histological and genetic alterations in respiratory epithelium remains to be established.
An improved understanding of premalignant bronchial epithelial cell biology is essential to identify reliable intermediate biomarkers for screening and chemoprevention. An increasingly better-established biological property of invasive lung carcinoma is angiogenesis, which is now understood to be a requirement for invasive tumor growth and metastasis (12) and has been identified as an independent prognostic variable in lung cancer (13 , 14) . Blood vessel formation in normal tissues is maintained at a static low level by a balance between angiogenesis activators and inhibitors. During tumorigenesis, alteration of this balance is thought to occur abruptly, and progenitor cells assume an "angiogenic phenotype," stimulating the formation of new blood vessels (15 , 16) . This sudden change in tumor progenitor cells is referred to as an "angiogenic switch" (17) . Although invasive tumors clearly may exhibit an angiogenic phenotype, the occurrence and timing of angiogenic switching in preinvasive lesions are not well delineated, particularly in the airways.
To better define morphological and genetic abnormalities of the airways during lung carcinogenesis, we performed both white light and fluorescence bronchoscopy on a population of current and ex-smokers previously shown to be at increased risk for dysplasia and lung cancer (18) . Fluorescence bronchoscopy is an investigational tool based on the observation that atypical or neoplastic epithelium emits a different spectrum of light from that of normal respiratory mucosa, and this difference can be exploited to identify atypical bronchial mucosa for molecular analysis (19 , 20) . It is currently being evaluated in clinical trials to identify premalignant bronchial epithelium and early lung cancer. In the course of these studies, we noted a microscopic lesion consisting of capillary-sized vascular projections occurring in association with highly proliferative metaplastic or dysplastic bronchial epithelium, ASD. ASD was absent from the airways of normal volunteer subjects but was frequently present in individuals at high risk for lung cancer. We determined that the lesion is associated with increased stromal MVD and allelic loss on chromosome 3p. ASD reflects a unique form of neoangiogenesis associated with dysplastic epithelium in the lower airways of individuals at high risk for lung cancer.
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Patients and Methods |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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Persons fulfilling at least one set of these sets of criteria underwent both white light bronchoscopy using a fiberoptic bronchoscope (Olympus BF20D; Olympus America Inc., Melville, NY) and fluorescence light bronchoscopy using a LIFE device (Xillix LIFE-Lung Fluorescence Endoscopy System; Xillix Technology Corp., Richmond, British Columbia, Canada). The same set of bronchoscopists performed all examinations. Biopsies were obtained from suspicious areas identified during either white light or fluorescent examinations as well as from two normal-appearing sites per patient.
Histopathology.
All biopsies were fixed in 10% buffered formalin and embedded in
paraffin wax. Paraffin blocks were sectioned according to a standard
protocol at 5 µm, and serial sections were mounted in consecutive
order on silane-coated glass slides (Superfrost-plus; Fisher
Scientific). Twelve slides containing eight sections/slide were
prepared from each paraffin block. Slides 4 and 9 were stained with H&E
and examined for histological diagnosis by light microsopy. The
remaining slides underwent immunohistochemical analysis or
microdissection for DNA extraction.
Epithelial abnormalities were classified according to recent WHO criteria as normal, reserve cell hyperplasia, squamous metaplasia, or dysplasia (low or high grade). The presence or absence of ASD as defined below was noted in each biopsy. Two examiners evaluated the specimens, and joint review and consultation resolved diagnostic disagreements.
Endothelial Cell Identification and MVD.
Endothelial cells were highlighted in tissue sections by
immunohistochemical staining using the endothelial marker CD-31. This
marker was used to confirm the presence of capillary cores in the ASD
lesions and to identify capillary vessels for MVD determinations. To
demonstrate this antigen, paraffin sections were stained with mouse
anti-CD-31 antibody (DAKO, Carpenteria, CA) using protease for
antigen retrieval and avidin-biotin/3,3'-diaminobenzidine (Ventana
Medical Systems, Tucson, AZ) for detection on the NEXUS automated
immunohistochemical stainer (Ventana Medical Systems).
Sections stained with anti-CD-31 were examined for MVD in the mucosa beneath normal epithelium, ASD, and other abnormal epithelium using the Image-Pro Plus image processing program (Media Cybernetics, Silver Spring, MD). Images of bronchial mucosa, usually to the level of smooth muscle or cartilage, were digitally captured using a x20 microscope objective. Cross-sectional mucosal areas were determined by manually tracing the region of interest on the digital image. Capillary-sized blood vessels within the area of interest were then counted, and MVD was expressed as microvessels/mm2.
Epithelial Cell Proliferation Rate.
Epithelial cell proliferation rates were estimated by
immunohistochemical staining for Ki-67, a nuclear antigen that is
expressed in G1, S phase, G2, and M phase of
the cell cycle but is absent in the G0 phase (22
, 23)
. Ki-67 immunostaining correlates well with thymidine
labeling index (24)
and has proven to be comparable to
mitotic count and flow cytometric analysis in defining tumor growth
fraction.
MIB-1, an anti-Ki-67 monoclonal antibody (Ref. 25 ); Immunotech Inc., Westbrook, ME), was used in an antigen retrieval procedure (Antigen Retrieval Citra; BioGenex, San Ramon, CA) to demonstrate Ki-67 in paraffin sections. A light hematoxylin counterstain was applied before viewing. Ki-67 growth fractions were calculated by counting the proportion of 400 epithelial cell nuclei that stained brown, regardless of intensity of staining, and multiplying by 100. Counts were performed in areas exhibiting ASD and in regions of histologically normal mucosa (when available).
Molecular Analysis.
For molecular analysis, slides containing ASD lesions were
deparaffinized and stained with H&E or methyl-green pyronine. We found
that although morphological detail was superior in H&E-stained
sections, amplification sometimes failed in DNA isolated from sections
stained in this manner. In such cases, sections were stained with
methyl-green pyronine, with successful results. Five µm
deparaffinized specimens were immersed in 100% ethanol for 5 min and
subsequently covered with a glycerol solution [95% glycerol from
Fisher Biotech (Fair Lawn, NJ)/5% PBS]. Epithelial cells were then
microdissected along with a representative stromal sample
(predominantly tissue leukocytes) from the same anatomic site using a
glass micropipette and a dissecting microscope. DNA was extracted from
microdissected cells by overnight proteinase K digestion at 55°C and
was isolated using the QIAmp Tissue Kit according to the
manufacturer’s instructions (Qiagen Inc., Valencia, CA). Subjects
enrolled in the study also submitted blood samples, and DNA was
isolated using a 341 GenePure Nucleic Acid Purification System (Applied
Biosystems, Foster City, CA).
3p Analysis.
Sites chosen for PCR amplification are highlighted in the ideogram of
the short arm of chromosome 3 (left side of Fig. 9
). These
regions have previously been shown to be homozygously deleted in lung
tumors or cell lines or are consistently deleted in premalignant
lesions and may contain putative oncogenes or tumor suppressor genes
(10
, 26) . Purified DNA samples underwent PCR to amplify
the following microsatellite markers: (a) 3p21.33,
D3S1611, D3S1298, and D3S1260; (b)
3p21.31, D3S2968, and D3S1573; (c)
3p14.2, D3S1300 and D3S1481; (d) 3p13,
AFM320, D3S1598, and 1284; and (e)
3p12.1, D3S1577, D3S1604, D3S1776, and D3S1274
[primer sequences were published previously in Ref.
(27)
].
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Chemiluminescence was used to detect regions of interest using the Phototope—Star Detection kit (New England Biolabs, Beverly, MA). In keeping with previously published work, LOH was determined by the complete absence or a decrease in visual intensity of at least 50% in one allele (10) . The amount of DNA in the microdissected samples was estimated by comparison with a known quantity of DNA from peripheral blood lymphocytes of the individual. Microsatellite instability was detected by a shift in the mobility of one or both alleles (8) . All determinations of loss were based on direct visual comparison with blood and stromal samples from the same patient. In situations where shadow bands existed above and/or below the principal allelic band, a common occurrence in microsatellite studies, the most intense band was considered the allele.
p53 Analysis.
DNA was analyzed for p53 mutation by single-strand
conformation polymorphism with direct sequencing, as described
previously (28)
. DNA was amplified for single-strand
conformation polymorphism after a proteinase K digestion, using six
nested primer sets encompassing exons 5A, 5B, 7, and 8
(28)
. These exons were chosen because of their association
with the large majority of p53 mutations. DNA from suspected
mutation sites was isolated and sequenced after amplification with
nonlabeled internal primers and gel purification. For sequencing, the
fmol DNA sequencing kit (Promega Corp., Madison, WI) was used
with autoradiography. DNA from cell lines with known p53 mutations and
water blanks were run as controls for all sequencing reactions.
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Results |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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Histopathology of ASD
Collections of capillary-sized blood vessels closely
juxtaposed to dysplastic epithelial cells were observed in biopsies
obtained during fluorescence bronchoscopy. Capillary loops frequently
projected upward into the bronchial mucosa, imparting a papillary
microscopic configuration to lesions (Fig. 1)
. These lesions were easily
recognizable in histological sections stained with H&E. The constituent
epithelium of the lesion was dysplastic to a variable degree as
indicated by: (a) overall increase in the cellularity of the
mucosa; (b) expansion of the basilar zone; (c)
variable progression of cell maturation from the basal to luminal
surfaces, frequently with flattening of epithelial cells in upper
layers and loss of superficial mucociliary cells; (d) nuclei
vertically oriented in the lower two-thirds of mucosa; (e)
increased nuclear:cytoplasmic ratio; (f) nuclear
angulations, grooves, lobulations, and asymmetry; (g)
frequent presence of nucleoli; and (h) occasional mitotic
figures, often above base of the mucosal epithelium. Thickened basement
membranes were a variable feature of ASD, with some lesions exhibiting
a marked increase in basement membrane thickness. ASD lesions could be
single or multiple.
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, along with two
representative cross-sectional planes. The three-dimensional image
confirms that ASD lesions represent projections of capillary loops into
the bronchial mucosa.
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confirmed the presence of endothelial
cells lining the capillaries in the cores of the papillary projections.
This immunostain also revealed that MVD immediately beneath ASD was
markedly increased in comparison to MVD beneath normal epithelium (Fig. 4)
. The mean MVD for ASD was
370/mm2 compared with 229/mm2 beneath normal
epithelium (P = 0.0003; two-tailed Student’s
t test). Other epithelial lesions without ASD were also
associated with an increase in MVD (415/mm2) in comparison
to normal epithelium (P = 0.00002; two-tailed
Student’s t test) but lacked intraepithelial capillary
loops, suggesting that ASD represents a qualitatively distinct,
histologically recognizable form of angiogenesis in airway mucosa.
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Immunohistochemistry for Ki-67 revealed a significantly elevated growth
fraction (n = 11, median = 35.1%, range =
7.7-68.9%). Histologically normal biopsies from this cohort were also
stained and had a significantly lower proliferative fraction
(n = 7, median = 1.5%, range = 0.7-3.5%).
When compared with normal epithelium, ASD lesions had a significantly
higher proliferation index (P = 0.00005; one-tailed
Student’s t test).
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, a
large majority (101 of 130; 78%) of ASD lesions were found at biopsy
sites judged to be abnormal by fluorescence bronchoscopy, and 59% of
the lesions were found in areas that were thought to be abnormal by
fluorescence but not by white light bronchoscopy.
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. Twelve percent (161 of 925) of
the biopsies from these smokers contained ASD. Multiple biopsies from
the same patient often displayed ASD. ASD was present in more that one
biopsy in 70% (38 of 54) of subjects with the lesions, and in 25 of
these multifocal cases (66%), ASD lesions were bilateral. In 11
subjects, a total of 20 sites containing ASD were rebiopsied 1 year
after the initial diagnosis. ASD was found to persist at 9 of these 20
sites (45%). No particular bronchial site was found to be
preferentially involved. ASD was not identified in 39 biopsies from 16
never smokers and 2 volunteers with minimal (0.5 and 3 pack-years)
smoking histories (P = 0.001; 
2 test).
The lesion occurred in 37% of males and 26% of females (Table 2)
evaluated; however, by
2 analysis, this difference was not statistically
different (P > 0.20). When clinical parameters in
high-risk subjects with ASD were compared with those in high-risk
subjects without ASD (Table 3)
, no
differences in age or tobacco exposure were found.
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contains a
representative gel illustrating LOH in an epithelial sample as compared
to the heterozygous stromal sample dissected from the same biopsy
specimen.
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contains a summary of the genetic
analysis. The extent of loss varied, with some subjects exhibiting LOH
only in one 3p region, whereas others had multiple areas of loss, as
illustrated by patient 11. Ten of 19 biopsies with ASD (53%) harbored
regions of 3p allelic loss. Subjects in our cohort without ASD had a
similar rate of LOH (66.7%). Thus, whereas a high incidence of LOH at
3p was observed at sites of ASD, LOH was not preferentially associated
with specific histological abnormalities, nor did more extensive loss
occur in ASD as compared to airway epithelium without ASD.
As indicated in Fig. 9
, a small number of cases were homozygous
for all primers in a particular 3p region and therefore uninformative.
Microsatellite instability, presenting as a shift (Fig. 8)
, was
observed in one specimen.
p53 Analysis.
No p53 mutations (codons 5, 7, and 8) were confirmed by
direct sequencing of the ASD biopsies. As we reported previously
(28)
, dysplastic bronchial epithelium from one patient did
have a distinct p53 mutation at several endobronchial sites, including
an ASD lesion, consisting of a G:C to T:A transversion in exon 7, codon
245 (GGC to TGC). To date, this p53 mutation is the only
p53 abnormality detected in our cohort with ASD.
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Discussion |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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The histopathology of a similar lesion was described as early as 1949 in an autopsy series from Finland (30) . No association with smoking was suspected at this time, which was before the landmark work of Wynder and Graham (31) linking tobacco smoking with lung cancer. In another autopsy study in 1963, a lesion described as "micropapillomatosis" was reported as an accompaniment of invasive lung carcinoma (32) . Dysplasia of bronchial epithelium in "micropapillomatosis" as well as a possible link between angiogenesis and preinvasive bronchial epithelial dysplasia was recognized by Muller, who also described the ultrastructure of these lesions, including documentation of their capillary cores (33) . However, these early studies have not been widely appreciated.
Currently, quantification of angiogenesis in human specimens relies on the counting of foci of vascular proliferation ("hot spots") in sections stained with antisera to vascular markers such as coagulation factor VIII and CD-31 or CD-34 antigens (15) . Such a method captures a snapshot of angiogenesis at a particular point in time but does not reflect the chronology of the process in the same tissue (34) . The frequent finding of thickened basement membranes in ASD suggests variable remodeling of the vascular extracellular matrix over an extended period of time.
Until recently, no means of locating such a microscopic lesion in the
lower airways has been available except for random biopsy. Our
experience with this lesion suggests that it may be recognized in
situ by fluorescence bronchoscopy. As summarized in Fig. 6
, 59%
of ASDs were abnormal under fluorescent light bronchoscopic examination
but normal under white light. Detection of preinvasive lesions by
fluorescence bronchoscopy is based on a reduction or quenching of the
fluorescent signal in suspicious lesions. The biochemical basis for
this phenomenon is unknown, but similar alterations in patterns of
fluorescence have been observed in early diagnosis of cervical cancer
(35)
. We believe that the increased or aberrant mucosal
microvascularity associated with ASD results in increased blood content
in the mucosa. This may result in quenching of the autofluorescent
signal and contribute to the recognition of these lesions in the lower
airway.
That the epithelium of ASD is abnormal is suggested not only by histological changes (dysplasia) but also by the increased proliferative rate of the epithelium and the presence of allelic loss in microdissected epithelial cells. The proliferative rate of normal respiratory epithelium, as measured by Ki-67 labeling index, is low (36, 37, 38) . In this series, in histologically normal epithelium, the mean Ki-67 labeling rate was 1.8%, whereas that of ASD epithelium was 38% (P < 0.00005). The significantly elevated proliferative rate in ASD may be a reaction to airway injury or may be due to endogenous proliferative stimuli resulting from mutation.
Genetic alteration of ASD epithelium is indicated by allelic losses at chromosome 3p. For the present study, alleles in five 3p regions were chosen for study. These regions were previously shown to have undergone frequent heterozygous or homozygous loss in tumors and, in some cases, in preneoplastic epithelium as well. Sites of possible tumor suppressor genes are 3p14.2 and 3p21.31. The former region encompasses the most common inducible fragile site in the human genome, FRA3b, as well as the fragile histadine triad (FHIT) gene. The latter region, 3p21.31, is gene rich and includes the H. SemaIV gene (39) . LOH at 3p21.31 has been demonstrated in invasive lung tumors and preinvasive lesions (11) . Homozygous losses have also been found here in a limited number of small cell lung cancer cell lines (27 , 40) . Other 3p regions in which homozygous losses have been found in lung cancer cell lines include 3p21.33, 3p13, and 3p12.3. Primers for all five of these 3p regions were used to amplify the DNA from microdissected epithelial cells from ASD in the current study.
Our findings document a high frequency of allelic loss by ASD epithelium at one or more of the tested loci on 3p, with two-thirds of subjects having lost at least one allele at one or more bronchial sites. No locus was preferentially affected, and losses appeared to be randomly distributed among the loci tested. Expansion of a single mutant epithelial cell may have occurred in some cases, as suggested by the finding that 17% of subjects exhibited loss of the same allele at more than one site. These findings indicate that the epithelium of ASD, in addition to being histologically abnormal, is often genetically altered as well. The allelic loss, however, cannot be interpreted as specific for ASD because 3p LOH has been reported in smokers regardless of histological appearance of the mucosa (9 , 10) .
Point mutation constitutes a second mechanism for tumor suppressor gene inactivation in lung cancer. p53 is of particular interest in this regard because loss of wild-type p53 in Li-Fraumeni fibroblasts results in reduction of the angiogenesis inhibitor thrombospondin-1 and conversion to an angiogenic phenotype (41 , 42) . At least one report indicates that p53 overexpression may be associated with angiogenesis in lung cancer (14) , although this report has been contradicted by other studies using different endothelial markers to assess angiogenesis (43) . p53 mutation in preinvasive bronchial epithelium has only rarely been reported. One study documented an increase in the number of mutant p53 alleles in bronchial biopsies from the same site over a 9-month period (44) . Another has described the presence of a single p53 mutant clone distributed through broad areas of the respiratory tract in a high-risk smoker without lung cancer (28) . With these exceptions, there have been no reports of p53 mutation in preinvasive epithelium, and it is widely suspected that p53 mutation occurs mainly at a late stage of lung carcinogenesis. Our results support this suspicion because we found no p53 mutations in the epithelium of the ASD lesions we tested. This result also suggests that p53 mutation is not critical for conversion to an angiogenic phenotype, and the critical genetic alterations required for ASD formation remain to be determined.
ASD clearly indicates abnormal vascularization of the bronchial mucosa that is reflected by both increased microvascular density and aberrant morphology of bronchial capillary bed. Although microvascular density may be elevated in the stroma beneath epithelium exhibiting a range of histological abnormalities, the constellation of microscopic features defining ASD indicates a mechanistically distinct form of angiogenesis in airway mucosa. Possible mechanisms of angiogenesis in ASD may involve small populations of dysplastic squamous cells harboring premalignant mutations transmitting angiogenic signals over very short distances.
Formation of new blood vessels ("neovascularization") is crucial
for tumor cell growth beyond a certain restricted size and rate. Vessel
formation is mediated by a complex network of molecular signals
including both inducers and inhibitors of proliferation and migration
of endothelial cells (reviewed in Refs. 12
and 15, 16, 17
). Isolation and
characterization of proangiogenic endothelial growth factors [such as
vascular endothelial growth factor, fibroblast growth factors, and
certain CXC chemokines (45
, 46)
] and endogenous
angiogenesis inhibitors (thrombospondin-1, IFN-
/ß, platelet factor
4, angiostatin, and endostatin) and delineation of the role of the
extracellular matrix in neovascularization have provided potential
molecular mechanisms for blood vessel growth during tumorigenesis
(47)
.
The precise timing of angiogenic switching during lung carcinogenesis is not yet clear, but in locations more accessible than the lung, such as the cervix, there is evidence that switching to angiogenic phenotype occurs at a preinvasive stage (48) . Better definition of the morphological and molecular pathways involved in premalignant bronchial preneoplasia may lead to means of effective intervention in lung carcinogenesis before irreversible progression to invasive lung cancer takes place. At the present time, longitudinal studies are in progress to determine the long-term prognostic significance of ASD. The frequent occurrence of this lesion in high-risk smokers and in association with invasive carcinoma suggests that it may represent an important marker of lung cancer risk and may be a useful intermediate end point biomarker for chemoprevention studies. The demonstration that ASD occurs frequently in high-risk individuals suggests that antiangiogenic strategies may be effective in this subset of smokers at risk for lung cancer.
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FOOTNOTES |
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1 Supported by National Cancer Institute
Specialized Program of Research Excellence (SPORE) Grant CA 58187 and
National Cancer Institute Grant U01-CA85070 (Early Detection Research
Network). 
2 To whom requests for reprints should be
addressed, at Department of Pathology, Box B-216, 4200 East 9th Avenue,
Denver, CO 80262. Phone: (303) 315-1807; Fax: (303) 315-1835; E-mail: wilbur.franklin{at}uchsc.edu
3 The abbreviations used are: CIS, carcinoma
in situ; LOH, loss of heterozygosity; ASD, angiogenic
squamous dysplasia; MVD, microvessel density; LIFE, laser-induced
fluorescence emission; PBS, phosphate buffered saline.
Received 6/ 4/99; revised 1/ 4/00; accepted 1/18/00.
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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