HISTOLOGY OF SQUAMOUS DYSPLASIA

Because premalignant lesions and early carcinomas are not easily
recognized by white-light bronchoscopy, the stimulus for
evaluating the lower airways to identify lower airway neoplasia
by this method has not been great. Only recently has it
been possible to detect premalignant lesions using fluorescence
bronchoscopy as described in this volume and elsewhere. 13,14
This technical advance has engendered a need for a
reproducible and less descriptive classification of bronchial
premalignancy than that used in the earlier studies of Auerbach.
The WHO pathology panel has recognized this need and has
provided a suggested classification based largely on the earlier
work of Saccomanno and illustrated in Figure 22.2. The
classification is based on cellular changes that occur in the epithelium,
which consist of a transformation of bilayered mucociliary
epithelium to squamous epithelium that is associated
with varying degrees of alteration in nuclear irregularity and
mitotic activity. The classification includes seven categories
including histologically normal epithelium, basal cell hyperplasia
and squamous metaplasia, mild, moderate, and severe
dysplasia, and carcinoma in situ (CIS). Independent studies
have indicated that this classification is reproducible 15 and
may be used in clinical trials targeting premalignant changes
in the airways.
In addition to changes in the bronchial epithelium, changes
in the stromal support tissues have also been described. Potentially,
the most significant of these changes may be microscopic evidence
neoangiogenesis that has been referred to as angiogenic squamous
dysplasia (ASD). 16 ASD is characterized by the sprouting of capillaries
into dysplastic squamous mucosa (Fig. 22.3). These lesions
are frequently multifocal, may persist for several years at the same
bronchial sites and are preferentially associated with squamous
carcinoma. 17
Although areas of squamous dysplasia are now more readily
identified in the central airways, the risk posed by dysplastic
lesions discovered at bronchoscopy has not been quantified.
What is known is that CIS is associated with progression to
progress with the level of dysplasia. Exceptions to this rule
are the biomarkers that are associated with cell proliferation.
Generally, there is an increase in the level of expression of cell
proliferation markers with increasing histological grade. Cyclin
D1, 32,35,36 cyclin E, 37 PCNA, 38 Ki-67, 24,27,28 and MCM2 24,30
all increase with increasing grade of dysplasia, reflecting the increased
proliferative capacity of more severely dysplastic cells.
With the possible exception of proliferative immunohistochemical
biomarkers, no single change in immunohistochemically
demonstrable protein expression has to date added significantly
to the information that can be gleaned from conventional histological
examination.
Genetics of Preneoplasia Underlying the morphological
and immunohistochemical changes that occur in the
airways is a multistep sequence of molecular and chromosomal
events illustrated in Figures 22.4 and 22.5. The initial
event in lung carcinogenesis is the formation of DNA adducts,
the physical complexes between DNA, and the reactive
metabolites in tobacco smoke and industrial pollutants. 39–41
Among the most potent of the carcinogens are polycyclic
aromatic hydrocarbons (PAH), aromatic amines, and metals.
These compounds are largely metabolized to execrable
products by cytochrome p450 and glutathione S transferase
(GST). However, some small fraction of intermediates is
highly reactive with DNA and forms bulky adducts with
DNA in which the reactive metabolite is covalently bonded
to specific DNA bases. 40
DNA adducts activate complex DNA repair mechanisms,
which are not completely effective in removing adducts from
damaged DNA. Unrepaired DNA bases may be bypassed by
DNA polymerase, creating mutations that are transmitted to
daughter cells. Mutations formed in this way tend to favor
GC→TA transversions. Many of the genetic changes that ultimately
appear in lung carcinomas are thus thought to originate
from misrepaired DNA adducts.
Among these changes are allelic losses, easily demonstrated
by a simple molecular test based on the measurement of the
length of polymorphic tandem repeat sequences that exist
throughout the genome. In these tests, chromosomal loci that
normally harbor two different polymorphic alleles are assessed
for loss of one (loss of heterozygosity [LOH]) or both of these
alleles. Loss of both alleles (homozygous deletion) results in silencing
of the gene while loss of a single allele (heterozygous loss)
causes loss of gene expression if the retained allele is mutated or
inactivated by methylation. Loss of genes that are important in
controlling cell growth, apoptosis, or error DNA replication can
impart a malignant phenotype to lung cells and much effort has
been expended in trying to identify candidate tumor suppressor
genes in dysplastic epithelium and invasive tumors. 42
Allelic losses at loci throughout the genome have been
demonstrated in premalignant bronchial mucosa and detailed
consideration of specific allelic losses is reviewed elsewhere. 43,44
Several conclusions may be drawn from LOH studies performed
so far. First, many regions of allelic loss have been demonstrated
in smoking damaged airways but few studies have demonstrated
any effect of LOH on corresponding gene expression. Second,
many loci demonstrate loss from the earliest exposure to tobacco
smoke but allelic loss does not occur in individuals who have
never smoked. 45 Finally, no specific loss appears to be crucial
for progress to malignancy but rather it is the accumulation of
multiple losses that is most tied to malignant progression. 46
The molecular mechanism responsible for allelic loss is not
fully known. It has been suggested that bulky adducts resulting
from DNA oxidation could affect repair of double-stranded
DNA breaks and result in the recombination of homologous
recombination of DNA strands during the repair process.
Fluorescence in situ hybridization (FISH) studies have indicated
an increase rather than a decrease in gene copy number occurs at
sites of allelic loss, 47 suggesting that tumor cells harbor multiple
copies of the same allele and that gene dosage may in fact be increased.
Allelic loss may result in functional loss of protein only
when the retained allele is mutated or silenced by methylation.
A molecular property that distinguishes lung cancers and,
to a lesser extent, premalignant lesions from normal bronchial
cells is chromosomal instability, which is reflected as aneuploidy
in tumor and premalignant cells. Aneuploidy is first detected in
squamous dysplasia 48 and in one recent study, aneuploidy was
found in cultured bronchial epithelial of 26% of high-risk smokers.
49 In invasive lung carcinomas, aneuploidy is nearly universal
and involves multiple chromosomes. 50 Aneuploidy has been
found in lung cancer by classical cytogenetics methods 51 but is
more efficiently demonstrated by FISH. Numerical abnormalities
have been demonstrated in every chromosome by FISH. 52–54
New technologies such as spectral karyotyping (SKY)
(Fig. 22.5) and comparative genomic hybridization indicate that
not only are numerical chromosomal imbalances frequent but
also structural chromosomal abnormalities such as translocations
and amplifications are ubiquitous as well. 55 This high degree of
chromosomal instability may explain the extreme molecular and
cellular heterogeneity of lung cancers as well as their adaptability
and resilience in the face of chemotherapeutic treatment.
Patients with squamous as well as large and SCLC frequently
harbor dysplastic squamous lesions in the central airways
although the precise frequency in which this occurs is not
known. There is also increasing evidence of genomic and phenotypic
plasticity in invasive carcinomas, and tumors of mixed histological
type are a frequent finding in the lung suggesting that
tumor cells of many histological types could arise from a common
progenitor. Molecular evidence indicates that lung tumors
recapitulate ontological development, 56,57 suggesting that central
airway lesions of various histological types could represent
arrested development at various stages in the same progenitor
cells rather than origin from separate progenitor cell lineages.
Invasive Squamous Carcinoma of the Bronchus
Squamous carcinomas are in the most common of the central
airway tumors and are highly associated with smoking. 58
Invasive squamous tumors are characterized by extension of
malignant squamous cells beyond the basement membrane
of the airway lining. Approximately 29% of lung cancers
are of this histological type (Table 22.1). 59 The diagnosis of
squamous carcinoma and indeed of all non–small cell tumors
has taken new importance with the recognition that new targeted
agents may differentially affect non–small cell subtypes
and it is therefore more important to recognize and report
squamous lesions than in the past.