Molecular Prognostication of Lung Cancer

At present, the best prognostic indicator of long-term survival
for patients with lung cancer is tumor stage. The recent proposed
revisions to the TNM Classification of Malignant Tumours
by the International Association for the Study of Lung Cancer
(IASLC) are an analysis of 67,725 cases of non–small cell lung
cancer (NSCLC) to further refine the prognostic accuracy of
tumor staging (see Chapter 30). 1 Regrettably, however, up to
30% of patients who undergo curative resection for stage I
lung cancer will have recurrence of their disease. Present conventional
wisdom is that the long-term survival in many cancers
may be increased if clinicians had the means to identify
and treat patients who would benefit from adjuvant therapy
that might not otherwise be indicated based on their initial
tumor stage. To this end, many investigators have examined
molecular and genetic factors that may influence tumor behavior
and therefore long-term prognosis.
A number of genetic alterations and abnormal expression of
several regulatory genes have been detected and described for lung
cancer. These alterations are caused by gene mutation, chromosomal
modification, epigenetic silencing, and deregulated messenger
RNA (mRNA). In addition, several studies have correlated
specific molecular genetic changes with clinical prognosis and survival
for patients with lung cancer. Currently, several clinical trials
are underway to further define patients’ molecular “signatures” in
an effort to predict both overall prognosis as well as response to
therapy for lung cancer (http://www.clinicaltrials.gov).
Early studies attemping to comment on prognostic variables
in lung cancer are summarized in a comprehensive and
systematic review of 887 studies, which identified 169 host- and
tumor-related molecular factors associated with prognosis in
lung cancer. 2 Several factors were significantly associated with
survival, independent of stage and reported in more than three
studies: p53, p21, Ki-67, and p185 gene status; serum cytokeratin
19 fragments; agryophilic nucleolar organizer region; and
markers of angiogenesis such as vascular endothelial growth factor
(VEGF) expression and vessel invasion. Other studies have
used molecular, immunohistochemical, and clinical–pathologic
markers to predict patient prognosis and outcomes. 3–5
The subsequent development of oligonucleotide and complementary
DNA (cDNA) arrays to analyze gene expression on
a larger scale has contributed significantly to the understanding
of molecular and genetic alterations in lung cancer. These
tools allow for the simultaneous analysis of literally thousands
of genes such that a genetic profile or signature can be constructed
for a particular patient or tumor. In addition to genetic
profiling, investigators have identified several proteomic (see
Chapter 9) and microRNA (miRNA) profiles for lung tumors.
These profiles are correlated to clinical behavior, thereby providing
prognostic information for patients with lung cancer.
INDIVIDUAL GENE ALTERATIONS AND
PATIENT SURVIVAL
K-ras Members of the ras gene family encode cell membraneassociated
G-proteins, which serve as mediators of signal transduction
for cellular proliferation (also reviewed in Chapter 5).
Up to 30% of NSCLCs are characterized by mutations in the
k-ras gene6,7; most of these mutations are found in adenocarcinomas
and are associated with a history of tobacco use. 8 The
most common mutation of k-ras is a G→T transversion in
codon 12 that results in constitutive activation and continuous
transmission of growth signals to the nucleus. Alterations in
k-ras appear to be early events in lung carcinogenesis, having
been observed in atypical alveolar hyperplasia lesions that are
thought by many to be precursors of lung adenocarcinomas. 9
The prognostic significance of k-ras mutations in lung cancer
remains controversial. Although many studies report an association
between decreased survival and worse prognosis in patients
whose tumors exhibit k-ras mutations, others, including
a metaanalysis of 881 cases, report no significant link between
ras mutation status and prognosis. 10–13
p53 The p53 tumor suppressor gene is mutated in more
than half of all human malignancies, and alterations in the
p53 gene are the most frequently found in human cancer (also
reviewed in Chapter 5). Approximately 50% of NSCLCs and
over 90% of small cell lung cancers (SCLC) harbor mutations
or deletions of the p53 gene. 10,14 Inactivation of p53 results
in diminished efficiency of DNA repair, derangements of cell
cycle regulation, and overall increased genomic instability. 15 In
the normal state, the p53 network is quiet and senescent. In
times of cellular injury or stress, however, the p53 network is
activated and its downstream effects include cell cycle regulation,
induction of apoptosis and DNA repair mechanisms.
A prospective study by Ahrendt et al. 16 demonstrated that
p53 gene mutations were independently predictive of decreased
survival in stage I tumors but not in stage II or III tumors.
Missense mutations were not significant for patient outcome.
However, p53 mutations that were truncating, structural, or
those abolishing DNA contact were associated with a poorer
overall patient outcome among all samples. The relationship
between p53 mutational status and adverse survival outcomes
has been corroborated by several other studies incorporating
NSCLC samples from all tumor stages. 17–25
Immunohistochemical studies of p53 have been less consistent.
In the largest studies examining p53 expression levels,
some authors have reported a correlation between abnormal
p53 expression and poor prognosis; however, others report no
statistically significant relationship. 3,26–33 Carbognani et al. 34
examined the role of p53 status in long-term survival following
resection of NSCLC. Using immunohistochemical analysis
of several prognostic markers, p53 status was the only independent
predictor of 10-year survival following resection of
adenocarcinoma. In another study, Tsao et al. 13 observed that
p53 protein overexpression was a marker of poor prognosis and
shorter overall survival. In addition, patients with tumors containing
wild-type p53 had a survival benefit from adjuvant chemotherapy
as compared to those with functionally aberrant p53
status. Despite the mixed evidence from immunohistochemical
studies, however, a metaanalysis of 56 studies was conducted to
further investigate the role of p53 alterations and lung cancer. 35
Abnormal p53 status was associated with decreased overall survival
in patients with NSCLC across all stages and in both
squamous cell and adenocarcinoma histologies.
Cell Cycle Regulation
Rb and p16 The retinoblastoma ( Rb ) susceptibility gene is a
tumor suppressor gene with a key role in human carcino genesis
(also reviewed in Chapter 5). The Rb gene is inactivated in
20% to 30% of NSCLCs and up to 90% of SCLC. 10 Despite
this, the effect of Rb mutation or abnormal expression on patient
prognosis is controversial, with most studies demonstrating
no significant relationship between Rb abnormalities and
survival. 27,36–38
However, Burke et al. 39 recently demonstrated that the
additive effect of concurrent abnormalities in either or both of
the Rb and p53 pathways was predictive of patient prognosis in
NSCLC. In this study, there was no association between patient
survival and isolated abnormalities of the Rb pathway proteins
pRb, cyclin D1, and p16 INK4A and p53 pathway proteins p53
and p21 Waf1 . However, certain combinations of abnormalities
were predictive of poor prognosis. These included concurrent
pRb negative status and cyclin D1 overexpression; concurrent
pRb negative, cyclin D1 overexpression, and p53 mutation;
concurrent cyclin D1 overexpression and p53 mutation.
The p16 INK4A gene, located on chromosome 9p21, is a
tumor suppressor gene that encodes a cyclin-dependent kinase
(CDK) inhibitor (see also Chapter 14). Normally, p16 binds to
the cyclin D/CDK4/6 complexes to inhibit phosphorylation of
the Rb protein, thereby inhibiting G1→S progression. In a recent
analysis of tumors from patients with histologically proven
N2 NSCLCs, the immunohistologic presence of both p16 and
p21 protein correlated with improved long-term survival. 40
Similarly, dysfunctional or absent p16 expression can result
in unchecked progression through the cell cycle. p16 plays
a prominent role in NSCLC; inactivation is present in 40%
to 70% of NSCLCs. Mechanisms of p16 inactivation include
point mutations or deletions in coding regions, as well as epigenetic
silencing by hypermethylation of the gene promoter
cytosine-guanine-phosphate (CpG) island. Alteration and inactivation
of p16 are associated with a number of clinical correlates
in NSCLC, including metastases, poor prognosis and
overall decreased survival. 41–44
The cyclins, p21 WAF1/CIP1 , and p27 Other cell cycle regulatory
genes of interest include cyclin D1, cyclin E, cyclin B1,
p21 WAF1/CIP1 , and p27. Cyclin D1 plays a role in cell cycle
regulation by allowing transition from G1 to S phase. Although
overexpression of cyclin D1 occurs in 25% to 47% of NSCLC,
its prognostic effects are somewhat controversial. In some studies,
overexpression has been correlated with the presence of
lymph node metastasis, advanced pathologic stage, and shorter
overall survival. 38,45 However, other investigators have reported
favorable outcomes associated overexpression of cyclin D1. 5,46
Cyclin E helps to regulate entry into the S phase of the cell
cycle by formation of a complex with CDK2 and subsequent
phosphorylation of pRb. High levels of cyclin E expression in
NSCLC are found in up to 53% of NSCLCs, and have been
correlated with tumor invasion, unfavorable prognosis, and
decreased patient survival. 47,48 The cyclin B1/CDC2 complex
regulates the G2-M phase checkpoint of the cell cycle. In early
stage NSCLCs, overexpression of cyclin B1 occurs more commonly
in tumors of squamous histology, and high levels of expression
have been linked to shorter survival. 49
p21 and p27 belong to the Cip/Kip family of CDK
inhibitors, which bind to and inactivate CDKs in times of
cellular stress, hypoxia, DNA damage, and in response to
growth inhibitory signals (also reviewed in Chapters 5 and
14). p21 WAF1/CIP1 can inhibit cell cycle progression at multiple
sites. Early in G1, p21 WAF1/CIP1 binds to the cyclin
D/CDK4 and cyclin E/CDK2 complexes. Prior to transition
from S phase to G2, p21 WAF1/CIP1 can inhibit the cyclin
A/CDK2 complex. Although some authors determined that
p21 expression was associated with improved survival, others
found no relationship. 50–52 p27 Kip1 interacts with both cyclin
D1 and cyclin E to regulate the cell cycle. Several studies have
employed immunohistochemical techniques to determine
p27 expression; decreased levels of p27 expression have been
uniformly correlated with poor prognosis in NSCLC. 53–55
Protein Kinases
EGFR The epidermal growth factor receptor (EGFR) family
(also reviewed in Chapters 5 and 49) includes a group of tyrosine
kinases whose activation results in a cascade of downstream
signals that ultimately enhance cellular proliferation,
tumor cell motility and angiogenesis, and decrease apoptosis. 56
Although EGFR is overexpressed in many epithelial cancers, including
40% to 80% of all NSCLCs, these aberrations are rare
in SCLC. 57 Downstream targets of EGFR activation include
the ras and raf pathways that directly regulate gene transcription
and cellular proliferation. Another gene targeted by EGFR activation
is the serine threonine kinase Akt , which acts as a key regulator
of cellular survival through suppression of apoptosis. 58
EGFR mutations in lung cancer are associated with nonsmokers,
women, patients from East Asian countries, adenocarcinoma
histology, and, specifically, bronchoalveolar subtype. 59–63
In addition, tumors with k-ras mutations (associated with tobacco
exposure) and those with EGFR mutations appear to be mutually
exclusive. 60,63 It was initially thought that EGFR tyrosine kinase
inhibitors such as erlotinib and gefitinib might revolutionize the
treatment of patients with overexpression of EGFR in NSCLC.
However, clinical studies have demonstrated significant responses
in only specific subsets of patients, limiting gefitinib to use as a
second- or third-line agent and erlotinib as a first-line agent specifically
for elderly patients and those with EGFR mutations. 64,65
The impact of EGFR mutation and overexpression on
lymph node metastasis, patient prognosis, and overall survival is
controversial. Gene amplification often occurs with EGFR overexpression
(as opposed to transcriptional or translational modification),
and this has been associated with lymph node metastasis
and advanced pathologic stage. 66 Although many have found
that EGFR mutation and overexpression correlates with worse
survival in NSCLC, other studies report no significant association
between the two. 59,63 A recent metaanalysis of 16 studies
found that immunohistologic expression of EGFR does not correlate
with overall prognosis in patients with NSCLC. 67
ErbB2/HER-2/neu Another member of the protein kinase
gene family is ErbB2/Her2/neu . Screening studies for mutations
in the kinase domain of ErbB2/Her2/neu in NSCLCs
have revealed that mutations in squamous cell carcinomas are
rare, but found in approximately 10% to 30% of adenocarcinomas.
68–70 As with mutations of EGFR, mutations of ErbB2/
Her2/neu are more common in nonsmokers than in smokers. 63
ErbB2/Her2/neu overexpression has been associated with early
tumor recurrence, chemotherapeutic drug resistance, poorer
prognosis, and overall shorter survival time. 71–74
Angiogenesis and Growth Factors (see also
Chapters 8 and 48) For tumors to grow, they must obtain oxygen
and nutrients. Tumors greater than 1 mm in size cannot
depend on simple diffusion and therefore must create a vascular
supply to meet these metabolic demands. VEGF is a potent
growth factor for endothelial cells, promoting angiogenesis by
increasing vascular permeability and stimulating endothelial
cell proliferation. The VEGF receptors, VEGFR-1, -2, and -3,
are tyrosine kinases. VEGF expression has been demonstrated
in NSCLCs and is stimulated by tissue hypoxia, other growth
factors, and cytokines. 75,76
The presence of VEGF in NSCLC tumors of all stages has
been uniformly correlated with poorer prognosis and impaired
survival. 77 Bevacizumab (Avastin) is a humanized monoclonal
antibody that binds to circulating VEGF and inhibits its interaction
with the VEGF receptors. The Eastern Cooperative
Oncology Group (ECOG) phase III trial E4599 demonstrated
an overall survival benefit for patients with advanced stage adenocarcinoma
who received bevacizumab in addition to paclitaxel
and carboplatin. 78 Another phase III trial, the European
AVAstin in Lung cancer (AVAiL) trial, demonstrated a favorable
progression-free survival for patients with nonsquamous
NSCLC receiving bevacizumab in addition to cisplantin and
gemcitabine. 79 In addition to other studies of bevacizumab in
NSCLC, several multitargeted tyrosine kinase inhibitors are
under clinical investigation. Targets of interest include several
VEGF receptors, EGFR, platelet-derived growth factor
(PDGF), raf, and kit.
Interleukin-8 (IL-8) also has angiogenic properties in
NSCLC. 80 IL-8 expression in tumors has been correlated not
only with angiogenesis and microvessel density, but also with
advanced stage, lymph node metastasis and overall patient
prognosis. 81 Other growth factors of interest include PDGF
and basic fibroblast growth factor (bFGF). PDGF increases
DNA synthesis, tumor growth, and endothelial cell migration;
it has been correlated with decreased 5-year survival for
patients with resected primary lung adenocarcinomas. 77,82
FGF2 stimulates tumor growth and angiogenesis, and in
vitro studies have established a synergistic effect of FGF2
and PDGF. 83
The Matrix Metalloproteinase Family The matrix
metalloproteinase (MMP) family is a group of proteolytic
enzymes associated with degradation of extracellular matrix
and penetration of basement membranes, two key elements
in the metastasis of tumors. MMP-2 (also known as gelatinase
A) has been associated with lymphatic and vascular
invasion of NSCLC. 84 Overexpression of MMP-2, as measured
by immunohistochemical analysis, has been identified
as a negative prognostic factor in lung cancer survival. 85
Similarly, differential levels of MMP-7 expression were
found between resected squamous cell carcinomas and
adenocarcinomas, with higher levels in the squamous cell
carcinomas. 86 MMP-7- positive status was significantly associated
with poor prognosis and shorter overall survival.
In contrast, the data regarding MMP-9 are controversial.
Although some studies suggest a negative prognostic influence
of MMP-9, others have found no significant relationship
between the two. 87–89
Recently, Sienel et al. 90 described a role for the extracellular
matrix metalloproteinase inducer (EMMPRIN) in determining
prognosis for lung adenocarcinoma. EMMPRIN is a
transmembrane glycoprotein that has been shown to stimulate
synthesis of several MMPs, including MMP-1, -2, -3, and -9.
EMMPRIN expression was determined in a cohort of NSCLCs
using immunohistochemical staining, and a score was assigned
to each specimen. Furthermore, investigators recorded either a
membranous or cytoplasmic pattern of staining. For patients
with adenocarcinoma, a membranous staining pattern was independently
associated with poor prognosis, defined as either
local recurrence or distant metastasis. These relationships were
not significant for other histologic subtypes.
Maspin is a member of the serpin (serine protease inhibitor)
family and has been shown to be a suppressor of tumor
growth and metastasis in several types of tumors. Maspin can
inhibit invasion and metastasis of malignancies, although direct
evidence of the clinicopathologic significance of cytoplasmic relative
to nuclear expression is limited. Cytoplasmic and nuclear
expression patterns of maspin are involved in the cellular differentiation
of normal lung tissue and the histogenesis of different
lung carcinomas. The cytoplasmic maspin may play an important
role in lung carcinomas by regulating apoptosis and thus
is a favorable prognostic marker for AD patients, whereas the
nuclear location may be linked to promotion of angiogenesis.
Immunohistochemistry reveals that maspin expression is virtually
universal in NSCLC, but squamous cell carcinoma show almost
exclusively a combined nuclear-cytosolic stain. In contrast,
nuclear maspin, but not combined nuclear-cytoplasmic maspin,
significantly correlates with low histological grade, lower proliferative
rate, absence of invasion, and negative p53 stain in
ACa. Nuclear localization of maspin may thus stratify subtypes
of NSCLC with favorable clinical–pathological features.