Download clinical review molecular markers of head and neck squamous cell

CLINICAL REVIEW
Mark K. Wax, MD, Section Editor
MOLECULAR MARKERS OF HEAD AND NECK SQUAMOUS
CELL CARCINOMA: PROMISING SIGNS IN NEED OF
PROSPECTIVE EVALUATION
Phillipe Lothaire, MD,1 Evandro de Azambuja, MD, MSC,2 Didier Dequanter, MD,1 Yassine Lalami, MD,2
Christos Sotiriou, MD, PhD,2 Guy Andry, MD,1 Gilberto Castro Jr, MD, MSC,2 Ahmad Awada, MD, PhD2
1
Department of Surgery, Institut Jules Bordet, Brussels, Belgium
Translational Unit of Medical Oncology Clinic, Institut Jules Bordet, Boulevard de Waterloo, 125,
1000 Brussels, Belgium. E-mail: [email protected]
2
Accepted 21 July 2005
Published online 11 November 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/hed.20326
Abstract: Background. The aim of this article is to review
recent developments in the biological understanding of head
and neck squamous cell carcinomas.
Methods and Results. We describe the markers according
to their function and their prognostic or predictive roles. Some
associations can be found between molecular markers and invasiveness, aggressiveness, degree of differentiation, and tumor
stage, but only a few clinical studies have shown an impact on
prognosis. In addition, despite an increasing number of articles
relating to this topic, the small number of patients included in the
studies reported reduces the clinical implications of these
results. Few studies applied a more comprehensive molecular
analysis approach, such as DNA microarrays or differential expression profiling by polymerase chain reaction, to identify a
combination of markers that could be more informative than a
single molecular marker.
Conclusion. Some progress has been made with respect to
molecular markers and head and neck cancers. Translational
Correspondence to: A. Awada
The first two authors have contributed equally to this article.
B 2005 Wiley Periodicals, Inc.
256
Molecular Markers in Head and Neck Cancer
and prospective, hypothesis-driven research must proceed with
sufficient rigor to facilitate the clinical applicability of such results.
A 2005 Wiley Periodicals, Inc. Head Neck 28: 256 – 269, 2006
Keywords: head and neck cancer; molecular markers; proliferative markers; squamous cell carcinoma; EGFR
H ead and neck squamous cell carcinoma
(HNSCC) is the fifth most common cancer worldwide with a global yearly incidence of 780,000 new
cases.1 Despite aggressive and multidisciplinary
treatment approaches, including preoperative or
postoperative chemotherapy and/or radiotherapy
with reconstructive surgery, there has been no
significant improvement in 5-year survival over
the past 20 years. Treatment failures still occur in
the form of locoregional recurrence, distant metastases, and/or second primary tumors.2 – 4
Currently, treatment strategies rely on clinical, radiologic, and histopathologic parameters to
determine the stage of the disease. It is widely
accepted that the presence of lymph node metasHEAD & NECK
March 2006
tases is the most adverse independent prognostic
factor in HNSCC. Furthermore, tumors might
differ according to their primary site, because the
behavior of an oral cancer is quite different from
that of a laryngeal one, for example.
Wide heterogeneity in terms of clinical outcome
and response to treatment exists even in patients
who are assigned to the same risk group. Thus,
much work is currently focused on the identification of better biological and clinical factors
that may serve as prognostic and predictive markers. The recent advances in basic research and
genomics have improved our understanding of the
biological basis of tumor development and progression. As a result, a variety of molecular tumor
markers characterized in the laboratory have been
studied in HNSCC for their potential to predict
disease outcome or response to therapy in patients.
With these developments, we hope in the near
future to be able to either predict patients at risk
for disease progression after standard therapy or,
for example, identify those who may benefit from
postoperative radiotherapy as well as those with
radioresistant tumors.5
In this article, we reviewed the literature on
the use of molecular biomarkers in HNSCC as a
potential prognostic and predictive tool using Pub
MED to find the articles. Our search was limited
to articles published after 1990 and written in
English. Only studies with multivariate analysis
of clinical outcome were reported.
Molecular tumor markers were allocated into
four groups according to their function: (1) tumor
growth; (2) tumor suppression; (3) immune re-
sponse; and (4) angiogenesis, tumor invasion, and
metastatic potential.
MOLECULAR MARKERS INVOLVED IN TUMOR
GROWTH, PROLIFERATION, AND APOPTOSIS
Epidermal Growth Factor Receptor Family (Epidermal Growth Factor Receptor, Her-2/neu, c-erbB 3-4).
The epithelial growth factor receptor is a transmembrane receptor that binds epidermal growth
factor (EGF), transforming growth factor (TGF)alpha, and other regulating proteins. Epidermal
growth factor receptor (EGFR) – mediated signals
result in a complex cascade of signaling pathways
that influence normal cellular proliferation and
differentiation and lead to strong mitogenic
activity.5 The EGFR family includes EGFR,
HER-2/neu (c-erbB-2), HER-3 (c-erbB-3), and
HER-4 (c-erbB-4).6 Overexpression of EGFR is
observed in 42% to 80% of studied HNSCCs.7
Until now, seven of eight trials showed in
multivariate analysis a poorer outcome for patients with EGFR overexpression, accounting for
756 patients (Table 1), than for those patients
without. Most of the trials used immunohistochemistry (IHC) as a method to analyze EGFR,
and two of them used polymerase chain reaction
(PCR). The trials reported different primary
tumor sites, numbers of patients (47– 140), and
follow-up periods (18 – 70 months).6,8 – 14 Five
trials report worst overall survival (OS). Conversely, only Wen et al10 found no association
between EGFR and outcome in 68 patients with
laryngeal carcinoma treated with radiation ther-
Table 1. Trials of epidermal growth factor receptor in head and neck squamous cell carcinoma.
Patients
Treatment
Median
follow-up
Method
of detection
Prognostic
value*
Ref.
NR
18 mo
IHC
IHC
OS
OS and RFS
(6)
(8)
Laryngeal carcinoma (n = 140)
Laryngeal carcinoma (n = 68)
Primary HNSCC (n = 91)
Oral cavity carcinoma (n = 110)
Surgery F RT F Chemotherapy
Chemotherapy
Surgery
RT
Surgery
RT
Surgery F RT F Chemotherapy
Surgery F RT
49 mo
70 mo (mean)
49 mo
NR
OS and RFS
Noy
DFS
PFS
(9)
(10)
(11)
(12)
Primary HNSCC (n = 59)
Primary HNSCC (n = 54)
Surgery F RT or Chemotherapy + RT
Surgery (adjuvant therapy in some of them)
NR
26 mo
IHC
IHC
IHC
DNA PCR for
erbB-1/2
ELISA
RT-PCR
OS (2-y)
OCSS
(13)
(14)
Oral cavity carcinoma (n = 47)
Primary HNSCC (n = 109)
Abbreviations: RT, radiotherapy; NR, not reported; IHC, immunohistochemistry; OS, overall survival; HNSCC, head and neck squamous cell carcinoma;
RFS, relapse-free survival; DFS, disease-free survival; PCR, polymerase chain reaction; PFS, progression-free survival; ELISA, enzyme-linked
immunosorbent assay; RT-PCR, reverse transcriptase-PCR; OCSS, overall cancer specific survival.
*Multivariate analysis: The expression of the marker predicts worse outcome.
yOnly univariate analysis was performed.
Molecular Markers in Head and Neck Cancer
HEAD & NECK
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257
apy (RT). This study had the longest follow-up
period (mean, 70 months), and only a univariate
analysis was performed.
In patients treated with cisplatin-based chemotherapy for recurrent head and neck cancer,
positive staining for c-erB-2 was correlated with
shorter progression-free survival and OS ( p =
.023). Importantly, worse OS was also seen in
patients with c-erB-2+ who were treated with
cisplatin and paclitaxel ( p = .027), suggesting that
this overexpression may be associated with
chemotherapy resistance in these patients.15
EGFR seems to be strongly correlated with
worse prognosis in HNSCC in patients treated
with either chemotherapy or RT. A better understanding of its signaling pathways may improve
the treatment results of patients in this setting.
Cyclins are very important during the
cell cycle and are divided in to four types: A, B, D,
and E. Cyclin D1 (also known as PRAD1) is a
protooncogene that responds to extracellular
mitogens and is a controller of G1 phase progression through the cell cycle.5 The most commonly reported alteration of cyclin D1 is gene
amplification (11q13). This results in the expression of a structurally normal protein, but at abnormally high levels, which leads the cell to a
state of uncontrolled proliferation.
Cyclin D1 may be amplified in 30% to 50% of
laryngeal carcinomas, and its deregulation may
increase the aggressiveness of certain cancers.5
Capaccio et al16 found in 32 HNSCCs that cyclin
D1 expression is significantly correlated with
tumor extension and advanced clinical stage ( p =
.002 and p = .001, respectively).
Five of seven studies analyzed cyclin D1
using the IHC method, and Southern blot and
DNA amplification were used in other two
Cyclin D1.
(Table 2).17 – 23 The shortest follow-up period was
at least 18 months and the longest was 60 months.
All patients (N = 812) were treated with surgery
with or without RT.
The prognostic significance of cyclin D1 remains controversial, although current data suggest a relationship between cyclin D1 overexpression/amplification and poor outcome. Five of seven
trials showed either worse disease-free survival
(DFS) or OS in patients with cyclin D1 overexpression/amplification than in patients without
this characteristic.17,19 – 22 Three of these trials
studied only patients with laryngeal carcinoma,
and one included only patients with anterior
tongue carcinomas.17,21,23 The largest trial with
282 patients failed to find a relationship between
cyclin D1 gene amplification and DFS or OS.18
Nevertheless, the evidence from five trials suggests a poor prognosis for patients with HNSCC
with cyclin D1 overexpression.
Ki67 is a monoclonal antibody that binds to a protein that is expressed
during the G1, S, and G2 phases of the cell cycle.
There is a correlation between the percentage
of Ki-67 – labeled cells at the time of the surgery
and the TNM stage.24 Tumors with a high
percentage level of Ki-67 – labeled cells have been
associated with more advanced TNM stage
disease.24 Several studies have also revealed
that cell proliferation in invasive tumors measured by Ki-67 highly correlated with histologic
grading in human oral squamous cell carcinoma
(OSCC).25 – 27 In addition, a high percentage of
immunolabeled cancer cells for Ki-67 has been
significantly associated with neck metastases and
inversely correlated with the degree of cancer
cell differentiation.28
Ki67 (MIB-1) Antigen.
Table 2. Trials of cyclin D1 in head and neck squamous cell carcinoma.
Patients
Laryngeal carcinoma (n = 51)
Primary HNSCC (n = 282)
Primary HNSCC (n = 75)
Primary HNSCC (n = 45)
Laryngeal carcinoma (n = 149)
Anterior tongue carcinoma (n = 148)
Laryngeal carcinoma (n = 62)
Treatment
Median follow-up
Method of detection
Prognostic value*
Ref.
Surgery
Surgery F RT
Surgery F RT F
Chemotherapy
Surgery
Surgery
Surgery F RT
Surgery F RT
29 mo
28 mo
At least 18 mo
Southern blot analysis
DNA amplification of 11q13
IHC
OS
No
OS
(17)
(18)
(19)
44.5 mo
60 mo
57 mo (mean)
More than 3 y
IHC and PCR
IHC
IHC
IHC
5-y survival
DFS
OS
No
(20)
(21)
(22)
(23)
Abbreviations: OS, overall survival; HNSCC, head and neck squamous cell carcinoma; RT, radiotherapy; IHC, immunohistochemistry; PCR, polymerase
chain reaction; DFS, disease-free survival.
*Multivariate analysis: the expression of the marker predicts worse outcome.
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Molecular Markers in Head and Neck Cancer
HEAD & NECK
March 2006
Table 3. Trials of Ki-67 in head and neck squamous cell carcinoma.
Treatment
Median
follow-up
Method of
detection
Prognostic value*
Ref.
RT
Chemotherapy + RT
Chemotherapy + RT
RT
31 mo
46.9 mo
At 4 y
48 mo
IHC
IHC (MIB-1)
IHC
IHC
Ki67 > 20% better LC
No
OS
LCy
(29)
(30)
(31)
(32)
Patients
Primary
Primary
Primary
Primary
HNSCC
HNSCC
HNSCC
HNSCC
(n
(n
(n
(n
=
=
=
=
101)
111)
105)
304)
Abbreviations: HNSCC, head and neck squamous cell carcinoma; RT, radiotherapy; IHC, immunohistochemistry; LC, local control; OS, overall survival.
*Multivariate analysis: High Ki-67 tumors predict worse outcome.
yFor Ki67 < 20% + p53 z 10%.
Four studies had used IHC for Ki-67 evaluation, with a total of 621 patients, a minimum
follow-up of 31 months, and with controversial
results (Table 3).29 – 32
Few studies investigated the role of Ki-67 in
either DFS or OS. One of these studies showed no
prognostic significance in multivariate analysis.30
It seems that patients with high levels of Ki-67
have better local control than those patients with
low levels.29,32 Curiously, Ki-67 positivity predicted a worse OS in 105 primary HNSCC.31 Because of the scarce and conflicting data, no clear
conclusions can be made for the use of Ki-67 in
clinical practice.
Furthermore, in terms of prediction, two recent studies showed that highly proliferating
tumors (Ki-67 >20%) might respond better to
radiotherapy than slowly proliferative ones.29,32
These results are consistent with clinical experience, whereby rapidly proliferating tumors are
more sensitive to radiation therapy than those
growing slowly.24
Bcl-2. Bcl-2 is an antiapoptotic protein, being
part of the regulatory system that controls the cell
cycle and the induction of apoptosis. Alterations
in apoptosis-related genes may play an important
role in tumor progression and in the efficacy of
cytotoxic therapy and radiotherapy.33 However,
no significant correlation exists between the
expression level of the bcl-2 protein with histologic grade and the presence of neck metastases
in tongue carcinomas.34
Bcl-2 overexpression may be associated with
poor prognosis in some types of cancer (breast,
colon, and lung), favorable prognosis in others
(cervix, melanoma, bladder, and prostate), or no
significance in yet others (head and neck, endometrium, gastric, thyroid, and ovary).33
Two of six studies using IHC did not find any
association between Bcl-2 overexpression and
outcome (Table 4).35,36 Four of them found a better local control rate in bcl-2 – positive patients,
but an association with better OS was reported
in only one study, including 400 patients with
primary HNSCC enrolled in a randomized trial
with continuous hyperfractionated accelerated
radiation treatment or conventional RT.30,33,37,38
Conversely, Gallo et al37 reported that bcl-2 positivity was associated with worst OS and DFS in
71 primary HNSCCs. Currently, the relationship
between bcl-2 and outcome is not well known, and
Table 4. Trials of Bcl-2 in head and neck squamous cell carcinoma.
Treatment
Median
follow-up
Method of
detection
Prognostic value*
Ref.
Chemotherapy + RT
CHART or RT
Chemotherapy + RT
Pre- or postoperative therapy
RT
CHART (3 times day)
46.9 mo
At least 4 y
72 mo
At least 10 y
At least 5 y
19 mo
IHC
IHC
IHC
IHC
IHC
IHC
Better LC
Better LC and OS
No
No
Worse OS and DFS
Better LC
(30)
(33)
(35)
(36)
(37)
(38)
Patients
Primary HNSCC (n = 111)
Primary HNSCC (n = 400)
Advanced HNSCC (n = 79)
Laryngeal carcinoma (n = 83)
Primary HNSCC (n = 71)
Primary or recurrent HNSCC (n = 93)
Abbreviations: HNSCC, head and neck squamous cell carcinoma; RT, radiotherapy; IHC, immunohistochemistry; LC, local control; CHART, continuous
hyperfractionated accelerated radiation treatment; OS, overall survival; DFS, disease-free survival.
*Multivariate analysis: High Bel-2 tumors predict either better LC or worse outcome.
Molecular Markers in Head and Neck Cancer
HEAD & NECK
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259
no standardization of this method in these patients has been done.
Interestingly, in terms of prediction, a recent
study identified bcl-2 protein expression as an
independent marker to tumor response to induction platinum –fluorouracil-based chemotherapy
using two different cutoffs (5% and 10%) in 141
patients with HNSCC. However, the inclusion of
one bcl-2+ patient who did not respond to the
treatment resulted in a very high and imprecise
95% confidence interval upper limit (95% confidence interval, 2.3 – 170).39
Fas (or Apo-1/CD95) and its ligand
FasL are important mediators of apoptosis. The
Fas antigen is a cell surface protein that triggers
apoptosis on binding to the Fas ligand, largely
through activation of caspase 3, a downstream
protein of the apoptotic signal transduction cascade.40 Fas antigen is a member of the receptor
family of cell surface proteins, whereas FasL
belongs to the tumor necrosis factor family.41
Upregulation of functional FasL was reported
in human tumors such as the esophagus, lung,
brain, colon, and pancreas.41 Chen et al41 showed
downregulation of Fas and upregulation of FasL
expression in 24 OSCC. Sundelin et al42 observed
Fas expression in all of 24 moderately or welldifferentiated squamous cell carcinomas (SCCs)
of the tongue. Lora et al43 demonstrated that
FasL expression was negatively correlated with
the degree of differentiation and apoptosis in 19
OSCCs. There is evidence that FasL is fully
functional on the surface of HNSCC44 and that
serum levels of FasL are lower in patients with
HNSCC with active disease than in those patients
with no active disease or without tumors ( p <
.0183).45 Low serum levels and high levels of
FasL expression on the tumor suggest that the
tumor exerts systemic suppressive effects on immune cells, which may be partially mediated by
the Fas/FasL pathway.46 No Fas receptors were
detected in poorly differentiated tumors, which
might indicate a lack of activation of the apoptotic pathway.47
Although Fas is a very important mediator for
apoptosis, its role in prognosis in patients with
HNSCC still needs to be defined, and more
studies with large numbers of patients are needed
to reach a clear conclusion.
The lessons learned from all these molecular
markers involved in tumor growth, proliferation,
and apoptosis show us that they might have a role
Fas/FasL.
260
Molecular Markers in Head and Neck Cancer
on patient prognosis, in particular EGFR and
cyclin D1.
MOLECULAR MARKERS INVOLVED
IN TUMOR SUPPRESSION
p27 (Kip1) is a cyclin-dependent kinase
inhibitor (CDI), a member of the Cip/Kip group,
which negatively regulates the G1 phase progression of the cell cycle by binding to the cyclinE/
cyclin – dependent kinase-2 (CDK2) complex.48 p27
is activated in response to extracellular signals
such as serum deprivation, contact inhibition,
TFG-h,49 cyclic-AMP, and a growth inhibitory
drug (rapamycin).50 p27 is not a frequent target
of mutations predisposed to cancer and is considered a tumor suppressor gene.50
Overexpression of p27 using an adenovirus
vector system in several HNSCC cell lines has
shown a marked decrease in the proportion of
cells in the S phase, an increase in the G1 phase,
and a marked decrease in clonogenicity.51 In
contrast, high p27 degradation activity has been
found in highly invasive carcinoma cell lines
(MSCC-1).52
Reduced expression of p27 has been associated
with progression in precancerous lesions of the
larynx (leukoplasia), indicating the role of p27 as
a potent tumor suppressor gene.53 When 17
patients with oral epithelial dysplasia were
followed until progression to carcinoma, the high
p27 overexpression observed in the precancerous
lesions (88%) was replaced by low p27 overexpression in carcinomas (82%), and this reduction
may be involved in abnormal cell proliferation.54
Similar results are reported in 22 epithelial
dysplasia specimens in 70 OSCC cases. Moreover,
patients with metastases had lower levels of p27
compared with patients with tumors and no
metastases.55
In 31 untreated patients with HNSCC, a
significant correlation has also been found between low p27 (Kip1) expression and high-grade
tumors, advanced T stages, and positive cervical
nodal status. Furthermore, multivariate analysis
indicated that p27 (Kip1) expression was the most
significant predictor of OS among the different
variables assessed ( p = .03).48 Similarly, in 94 patients with oral tongue squamous cell carcinoma,
those with low p27 levels had shorter 5-year survival rates than patients with high levels ( p =
.024).50 In another study, no difference in survival
was observed in 93 patients with laryngeal and
oral cavity carcinoma.49
p27.
HEAD & NECK
March 2006
Clearly, p27 plays a very important role in the
cell cycle and carcinogenesis. Furthermore, low
expression of p27 confers a worse prognosis in
patients with HNSCC. However, it is not yet an
acceptable marker for clinical decision making.
In the late 1970s, p53 was discovered and
was supposed to be a critical modulator of the
cellular response to exogenous and endogenous
stress.56 Inactivation of one or more components
of the p53 network is an extremely common event
in human neoplasia. In HNSCC, disabling p53
occurs in a high proportion of cases by mutation
in the p53 gene. However, other mechanisms of
inactivation, such as the presence of human
papillomavirus (HPV) and molecular abnormalities in other components of this pathway, have
also been reported.56 In patients with HNSCC
who have HPV, this may occur by means of the
interaction of p53 with the E6 protein encoded by
so-called oncogenic HPV types, mainly HPV16 and
HPV18.56 Another mechanism involves mouse
double minute protein 2 (MDM2), which binds to
p53 and promotes the ubiquitination of the Cterminus of p53 and its subsequent degradation.56
In 69 patients with OSCC, expression levels of
MDM2 combined with p53 assessed by IHC have
been associated with tumor proliferation.57 In
addition, combined expression levels of p53/p21
and p53/mdm2/p21 have been reported as significantly correlated with lymph node metastases.57 Similarly, p53 expression was positively
associated with nodal involvement in 53 patients
with laryngeal and tongue SCC.58 Conversely,
p53 was not an independent predictor of metastases in 70 patients with advanced HNSCC.59
The frequent changes occurring in the p53
pathway in HNSCC imply that molecular genetic
and immunohistochemical analysis of this critical
tumor suppressor network may be of diagnostic
and prognostic use in the clinical management of
HNSCC.56 However, the prognostic value of p53
remains controversial. Teppo et al60 failed to find
any statistically significant association between
prognosis and p53, cell proliferation status, and
angiogenesis in a cohort of 100 patients with
laryngeal carcinoma.
In patients with HNSCC, many studies have
been conducted to evaluate p53 as a prognostic
factor. These studies had different numbers of
patients (from 43 – 304), follow-up periods (39 –
120 months), and treatments (surgery, RT, surgery ! RT, chemotherapy, chemotherapy + RT)
(Table 5).23,29,31,32,35,36,61 – 74 Multivariate analyp53.
Molecular Markers in Head and Neck Cancer
sis showed no prognostic value in five of 17 studies
using the IHC method to detect p53 mutation.35,61,65,67,68 Four of them had used PCR as
a method to detect this.66,69,72,73 A significantly
worse OS in patients with p53 overexpression was
found in nine trials.23,63,64,69 – 74 Some authors
failed to find a significant difference in OS, but
they found either a worse DFS or local recurrence
in patients overexpressing p53 than in those not
overexpressing p53.29,31,32,36,62,66,72 In laryngeal
carcinoma, worse OS was found in 62 patients,23
and worse DFS was reported in 83 patients.36 The
former had more than 3 years of follow-up, and the
latter had at least a 10-year follow-up. In patients
with hypopharyngeal SCC, no prognostic value
was found in 43 patients.61 In brief, our review
revealed that 15 of 20 studies showed worse
prognosis for those patients with a p53 mutation
than those without, and five of the studies were
unable to demonstrate this negative effect of the
p53 mutation. In the era of evidence-based medicine, a meta-analysis, which is level 1 evidencebased, should be undertaken to help us to determine the real role of p53 mutations in patients
with HNSCC.
With regard to p53 status and response to
chemotherapy in HNSCC, Bradford et al75 studied
the relationship of p53 mutations and sensitivity
to cisplatin in 23 HNSCC cell lines in vitro. They
showed that HNSCC cell lines harboring p53
mutations were more sensitive to cisplatin than
those cell lines with wild-type p53, suggesting
the role of the p53 mutation as a potential predictive marker for response to cisplatin-based
chemotherapy, although this difference was not
significant ( p = 0.4). In patients with HNSCC
treated with cisplatin and 5-fluorouracil in a
neoadjuvant setting, those whose disease was
unresponsive to treatment had a higher prevalence of p53 mutations than responders had ( p <
.04).76 Similarly, p53 expression was a strong
predictor of poor response in 236 patients with
HNSCC treated with induction chemotherapy
containing cisplatin and 5-fluorouracil.77 Concerning radiotherapy response, Kokoska et al78
studied 121 patients with glottal SCC treated
with exclusive radiotherapy, and no p53 predictive value was found.
A recent report has shown the utility of p53R2
mRNA expression, a new p53 target, as a marker
to predict tumor development and sensitivity to
radiochemotherapy in OSCC. p53R2 expression
was detected by in situ hybridization and was
more frequently expressed in dysplasias and SCC.
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261
Table 5. Trials of p53 in head and neck squamous cell carcinoma.
Patients
Laryngeal carcinoma (n = 62)
Primary HNSCC (n = 101)
Primary HNSCC (n = 105)
Primary HNSCC (n = 304)
Advanced HNSCC (n = 79)
Laryngeal carcinoma (n = 83)
Hypopharyngeal SCC (n = 43)
Primary HNSCC (n = 73)
Primary HNSCC (n = 69)
Oropharyngeal SCC (n = 106)
Primary HNSCC (n = 79)
Invasive HNSCC (n = 110)
Oral or oropharyngeal SCC (n = 69)
Primary HNSCC (n = 115)
Primary HNSCC (n = 39)
Primary HNSCC (n = 68)
Advanced HNSCC (n = 111)
Laryngeal, pharyngeal, and
oral cavity SCC (n = 114)
Invasive HNSCC (n = 190)
Advanced laryngeal or
pharyngeal SCC (n = 71)
Treatment
Surgery F RT
RT
Chemotherapy + RT
RT
Chemotherapy + RT
Preoperative or
postoperative therapy
RT ! Surgery
Surgery F RT
RT
Chemotherapy + RT
Surgery
Surgery ! RT
RT
Surgery
RT
RT
Surgery F RT
Surgery
RT
Surgery F RT
Chemotherapy
RT or chemotherapy
Neoadjuvant chemotherapy
Chemotherapy + RT
RT or surgery
Surgery
Surgery F RT
RT or chemotherapy
Chemotherapy !
Chemotherapy+RT or RT
Median
follow-up
Method of
detection
Prognostic value*
Ref.
>3 y
31 mo
At 4 y
48 mo
72 mo
At least 10 y
IHC
IHC
IHC
IHC
IHC
IHC
OS
LC
RFS and DSS
LC
No
DFS
(23)
(29)
(31)
(32)
(35)
(36)
43 mo
IHC
No
(61)
NR
NR
IHC
IHC
DFS
Time to SPM and TTF
(62)
(63)
At least 5 y
39 mo
12 – 55 mo
IHC
IHC
PCR
OS
No
LDFS
(64)
(65)
(66)
64 mo
45 mo
IHC
IHC
No
No
(67)
(68)
2 – 54 mo
At 24 mo
NR
After 60 mo
PCR
IHC
IHC
DGGE
and PCR
IHC and
PCR
DFS and OS
OS
DFS and OS
LC, DFS, DSS,
and OS
OS and DSM
after 550 days
(69)
(70)
(71)
(72)
IHC
OS
(74)
30 mo
67.4 mo
(73)
Abbreviations: RT, radiotherapy; IHC, immunohistochemistry; OS, overall survival; HNSCC, head and neck squamous cell carcinoma; LC, local control;
RFS, relapse-free survival; DSS, disease-specific survival; DFS, disease-free survival; NR, not reported; SPM, second primary malignancy; TTF, time to
failure; SCC, squamous cell carcinoma; PCR, polymerase chain reaction; LDFS, locoregional DFS; DGGE, denaturing gradient gel electrophoresis.
*Multivariate analysis: the expression of the marker predicts worse outcome.
Interestingly, p53R2 was significantly associated
with tumor size, lymph node metastases, and
histologic differentiation, as well as with complete
pathologic response to radiochemotherapy.79
The discovery of p53 autoantibodies (AAB) has
created a new serologic method to detect these
cells in HNSCC. Of note, 30% of patients who
were initially seen with p53 AAB did not correlate
with any histologic parameter,80 but a correlation
with the clinical course of the disease was found
in nine of 32 patients. Five of these nine are still
alive without any sign of tumor, and they show
constantly declining p53 AAB levels after primary
therapy. Three of these nine cancer patients died
during follow-up because of distant metastases,
one died without known cause, and all had a p53
AAB increase before they died.81
262
Molecular Markers in Head and Neck Cancer
p53 and p27 are the two most studied
molecular markers involved in tumor suppression in HNSCC. For p27, its low expression has
been related to carcinogenesis, and it is associated with a worse outcome. p53 was linked
to poor prognosis in most studies and has been
implicated in outcome for different tumors. Either a meta-analysis using individual patient
data or randomized prospective trials should
be conducted to better clarify its prognostic role
in HNSCC.
MOLECULAR MARKERS INVOLVED
IN TUMOR ANGIOGENESIS
Angiogenesis is the growth of new microvessels,
and this process depends on the motility, proliferation, and tube formation of endothelial
HEAD & NECK
March 2006
cells.82 Tumor vascularization is thus one of the
rate-limiting steps for tumor growth, invasion,
and metastases.
The vascular endothelial growth factor
(VEGF) family contains specific and highly potent
angiogenic proteins that act to increase vessel
permeability, endothelial cell growth, proliferation, migration, and differentiation. At least six
members of the VEGF family have been identified
so far, including VEGF-A/vascular permeability
factor, placenta growth factor, VEGF-B/VEGF –
related factor, VGFR-C/VEGF – related protein,
VEGF-D/c-fos – induced growth factor, and VEGFE. VEGF and basic fibroblast growth factor
(bFGF) are mitogenic and chemotactic for endothelial cells and are known to accumulate at the
site of angiogenesis in situ.82
Shang et al,83 in a small study of 31 patients
with OSCC, reported the circulating level of
VEGF and its relationship with clinicopathologic
features and prognosis. They showed that high
levels of serum VEGF correlated with node metastases ( p = .011) and clinical stage ( p = .024).
Enhanced expression of VEGF-A (isoforms 121
and 165) and VEGF-C may also predict the
presence of cervical lymph nodes metastases.84
Correlation between the intensity of VEGF expression and lymph node metastases was reported in 44 patients with primary oral SCC.85
Similarly, Teknos et al99 assessed serum VEGF
levels in a cohort of 183 patients wit HNSCC, as
well as in normal, healthy controls. They found
that serum VEGF levels were significantly elevated in patients with laryngeal carcinoma
compared with healthy controls. In addition, they
showed that high levels of VEGF were associated
with a supraglottal location of the tumor and with
node-positive disease. Finally, elevated pretreatment serum VEGF levels tended to indicate an
aggressive disease state and a short OS.
Neuchrist et al87 have investigated the expression of VEGFR3 and its ligand VEGF-C by
semiquantitative reverse transcriptase – PCR
(RT-PCR) in four HNSCC cell lines and six
HNSCC specimens and by IHC in another 18
HNSCC tumor specimens from 18 patients. They
failed to find a correlation between the expression
of VEGF-C and clinical parameters. In addition,
Ninck et al88 assessed the expression of several
angiogenic cytokines, including VEGF, bFGF,
platelet-derived growth factor (PDGF)-AB,
PDGF-BB, granulocyte colony-stimulating factor
(G-CSF), and granulocyte macrophage colonystimulating factor (GM-CSF) in HNSCC in vivo.
Molecular Markers in Head and Neck Cancer
They showed that tumors producing at least three
cytokines revealed a significantly poorer patient
prognosis, with a worse OS in univariate analysis,
than tumors that did not produce this. Moreover,
they suggested that VEGF and PDGF-AB might
play a key role in HNSCC and that the additional
secretion of G-CSF or GM-CSF might contribute
to a poor prognosis.
In 45 patients with oral SCC, those who were
VEGF positive had poorer OS than those who
were not.89 High levels of VEGF correlated with
more recurrence and a short disease-free interval.90 Similar results were found in patients with
high levels of VEGF and interleukin-8 (IL-8).91
Increased levels of b-FGF showed a significant
correlation with short time of locoregional control
in 26 patients with advanced HNSCC.92
In multivariate analysis, Salven et al 93
showed no association between VEGF and OS in
156 patients with HNSCC treated with surgery
and postoperative radiotherapy. Conversely, in 77
patients with oral or oropharyngeal SCC treated
with surgery and postoperative radiotherapy,
VEGF was the most significant predictor of poor
DFS (relative risk [RR], 2.75; 95% confidence
interval [CI], 1.30 – 5.79) and OS (RR, 3.53; 95%
CI, 1.75 – 7.13).94 In 60 patients with HNSCC, the
2-year DFS of patients with high VEGF levels
was significantly lower than that of patients with
low VEGF levels ( p = .01).95
In brief, VEGF has been highly correlated with
nodal invasion and worse prognosis in patients
with HNSCC. New biological agents targeting
tumor angiogenesis have been studied alone or in
combination with chemotherapy to achieve better
results in this patient population, and the results
of this research will be available soon.
Hypoxia-inducible factors (HIF1a and HIF2a)
are key proteins for response to hypoxic stimulus. The normal head and neck mucosa does
not show any reactivity for these two factors. In
human oral cancer cell lines, hypoxia is a mechanism that upregulates VEGF and HIF2a.96
In HNSCC, HIF1a and HIF2a overexpression
are significantly associated with high microvessel density, VEGF expression, incomplete response to chemotherapy, and a poor local relapsefree survival (RFS) and OS.97
Tumor angiogenesis can be quantified by
counting vessels per high-powered microscopic
fields. CD-31, the technique of choice to detect
microvessel density (MVD), failed to show any
correlation between tumor aggressiveness and
tumor angiogenesis in 19 patients with T1 oral
HEAD & NECK
March 2006
263
cavity SCC.98 Using the IHC technique, no
relationship between mean MVD and DFS or
OS was seen in 39 patients with HNSCC.69
Conversely, in 20 of 332 patients with laryngeal
SCC enrolled in the prospective trial ‘‘The Department of Veterans Affairs Cooperative Studies
Laryngeal Cancer Study #268’’, the mean vessel
count was significantly lower in patients who had
had chemoresponsive tumors ( p V .008). The most
vascular tumors, those greater than 1 SD above
the mean, had a poor OS ( p = .0345).86
Tumor microvessels are important for tumor
growth and survival, and the blockage of the
tumor vascular bed seems to be an attractive
approach in cancer treatment. Studies using biological agents, alone or in combination with chemotherapy, have already entered the clinic, and
it is hoped that the results will confirm the exciting preclinical data.
MOLECULAR MARKERS INVOLVED IN TUMOR
INVASION AND METASTATIC POTENTIAL
Matrix metalloproteinases (MMPs) are a family of
zinc metalloenzymes that are involved in extracellular matrix remodeling. The family of MMPs
is subdivided into collagenases, gelatinases, stromelysins, stromelysins-like MMP, membranetype MMPs, and new MMPs.100 It has been shown
that malignant cells are capable of using MMPs
to help break down the basement membrane and
degrade interstitial stroma, thus facilitating tumor invasion and/or metastases.4 The expression
of MMPs in OSCC has been reported to be correlated with tumor stage.4 However, several
published reports showed controversial results
of MMPs and their tissue endogenous inhibitors
(tissue inhibitors of metalloproteinases; TIMPs)
in relation to tumor progression, as well as to
the association between the plasma activity of
these enzymes and the known clinical and pathologic parameters of patients with head and
neck carcinoma.4,101
MMP-2 expression was significantly correlated with nodal status and OS in 106 patients
with advanced HNSCC.102 Molecular analysis of
surgical margins was done in 52 patients treated
with surgery with or without RT. Fourteen of 52
patients had recurrences, and 11 (79%) of these
patients had MMP-9 – positive margins. There
was a significant difference in the recurrence rate
between MMP-9 – positive and MMP-9– negative
margins (chi-square = 4.71; p = .03). A significant
correlation between patients with node-positive
264
Molecular Markers in Head and Neck Cancer
disease and overexpression of MMP-9 was observed ( p = .002).103
Serum levels of MMP-2 and MMP-9 were
collected in 86 patients with HNSCC and in
47 normal controls. Using a quantitative sandwich enzyme immunoassay technique, the MMP9 concentration was significantly higher in patients with HNSCC than in the controls ( p = .001)
and was correlated with advanced-stage disease ( p = .0449). No difference was found in
MMP-2 serum levels.104 MMP-3, MMP-8, and
MMP-9 serum concentrations had a statistically
significant difference in 73 patients with HNSCC
and 69 controls ( p < .001; p = .04; p = .001,
respectively). A significant correlation was found
between MMP-8 serum concentration and T
status ( p = .02), N status ( p < .001), and Union
Internationale Contre le Cancer (UICC) staging
( p = .03).100
Although a correlation exists between MMP
and cancer development in these studies, its use
is not recommended outside clinical trials. More
studies including a large number of patients are
necessary to define the real prognostic role of
MMP in HNSCC.
OTHER MOLECULAR MARKERS
The melanoma antigen (MAGE) genes encode
certain tumor-associated antigens recognized by
cytotoxic T lymphocytes.105 To identify immune
response targets in HNSCC, Kienstra et al106
studied the presence or absence of three antigens
identified to stimulate immune response, namely,
NY-ESO-1, MAGE-1, and MAGE-3, in 45 HNSCC
specimens by either RT-PCR or IHC. Interestingly, 27 of the 45 tumors assessed were positive
by RT-PCR for at least one of the antigens,
indicating a potential approach to immunotherapy for HNSCC. However, Kienstra et al were
unable to find any correlation between antigen
expression and patient demographics.
Finally, the binding of labeled galectin-3, an
endogenous lectin that reacts with glycan epitopes
of membrane and extracellular glycoproteins in
primary HNSCC (from the tonsil, base of the
tongue, and larynx), and lymph node metastases
has been assessed.107 It has been demonstrated
that galectin-3 has bound a nonproliferating pool
of tumor cells and can be localized with desmosomal proteins, suggesting that galactin-3 might
be a potential new tool for monitoring the degree
of cell differentiation in HNSCC. Unfortunately,
these markers are still not ready, and a better
HEAD & NECK
March 2006
understanding of their role as prognostic markers
in HNSCC is necessary.
Recently, a combination of circumstances,
including the advent of array-based technology
and progress in the human genome initiative, has
provided us with an ideal opportunity to begin
efforts aimed at performing comprehensive molecular and genetic profiling of human cancers
that will lead to a greater understanding of the
basic biology of HNSCC. Microarray technology is
a method used to interrogate 1000 to 40,000 genes
simultaneously. This technique permits the assessment not of individual genes but of clusters of
genes that are coordinately expressed to generate
‘‘fingerprints’’ of biological status.108 Comparative
genomic hybridization is a technique for detecting
segmental genomic alterations, in particular
chromosomal aberrations, giving us the possibility to detect genomic changes in malignant cells
in cases where aberrations are too complex to
study or when chromosomes are not available
at all.109,110
When comparing the gene expression profiles
of HNSCC and normal tissue, in the former,
altered expression levels of genes involved in the
control of cell growth and differentiation, angiogenesis, apoptosis, cell cycle, and signaling may
be seen.108
Following from this, Lemaire et al1 undertook
a large-scale differential display comparison of
hypopharyngeal SCC and histologically normal
tissue in a small series of patients. They identified 70 genes that exhibit a striking difference in
expression in tumor versus normal tissue. Thirtysix genes were detected with a greater than
twofold higher expression in tumor tissue than
in normal samples, and, inversely, 34 genes were
detected with a greater than twofold higher
expression in normal tissue than in tumor
samples. At the genome level, a series of differentially expressed genes cluster at 12p12-13 and
1q21, two hotspots of genome disruption. The
known genes share functional relationships in
keratinocyte differentiation, angiogenesis, immunology, detoxification, and cell surface receptors.
The differentially expressed genes that were
identified are potential new markers and therapeutic targets.
Patients with hypopharyngeal SCC have six
overexpressed genes (EIF4G1, DVL3, EPHB4,
MCM7, BRMS1, and SART1) and, from the
comparison of patients with nonaggressive and
aggressive tumors (without or with clinical evidence of metastases 3 years after surgery), it was
Molecular Markers in Head and Neck Cancer
found that 164 different expressed genes are
probably involved in the acquisition of metastatic
potential.111 From a study of nine patients with
HNSCC, gene expression changes were reproducibly observed in 227 genes representing previously identified chemokines, tumor suppressors,
differentiation markers, matrix molecules, membrane receptors, and transcription factors that
correlated with neoplasia, including 46 previously
uncharacterized genes.112
An analysis of 45 primary HNSCC by comparative genomic hybridization revealed significant correlations between the gain of 3q25-27 and
deletion of 22q with reduced disease-specific
survival. In addition, the gain of 17q and 20q,
deletion of 19p and 22q, and amplification of
11q13 were significantly associated with reduced
disease-free survival. A Cox proportional hazard
regression model identified the deletion of 22q as
an independent prognostic marker.113 Belbin
et al114 studied a set of 9216 sequence-verified
human IMAGE cDNA clones in 17 patients with
HNSCC treated with surgery. Two genotypic
groups were identified, and a trend to better
2-year cause-specific survival in the second group
was found (56% vs 100%; p = .057).
Smad2 protein was expressed in 99% of 170
HNSCC specimens. The activated form of Smad2,
pSmad2, was expressed in 86% of these tumors,
indicating their ability to survive and proliferate
despite the presence of bioactive TGF-h within
the tissue microenvironment.115
Another study suggested that the development
of cisplatinum-resistant phenotypes in head and
neck cancer is accompanied by alterations of gene
expression, including a glycoprotein hormone and
membrane proteins. These gene products might
be new molecular markers for resistance to cisplatinum. They demonstrate an upregulation of
GPHa in cisplatinum-resistant HNSCC lines and
two downregulated genes, human folate receptor
and L6 antigen.116
Using Atlas human cancer 1.2 cDNA array in
1187 tumor-related genes in either radioresistant
or radiosensitive tumors, 60 tumor-related genes
were selected as a predictors of radiation response of HNSCC.117 According to a very recent
literature review, this is a powerful method that
has an immense potential to help us understand
the important genetic changes in HNSCC.118
These high-throughput techniques are promising; however, they are not ready to be used in
the clinic. In the near future, tailored treatment
may become an option for patients with HNSCC,
HEAD & NECK
March 2006
265
which would avoid excessive toxicities from overtreatment.
CONCLUSION
Head and neck cancers are heterogeneous in
location as well as in their biological or clinicopathologic behavior. After the enthusiasm surrounding p53, many biomarkers have been identified and are now available. However, despite
advances in new techniques, those markers
present limited interest for the clinician so far
and are not able to provide firm conclusions about
their value in screening and as prognostic and
predictive factors.
The trials undertaken so far are also too
heterogeneous to provide reliable data, as is the
case with p53. Some studies have demonstrated
an association between p53 abnormalities and
poor outcome, but other large studies have failed
to demonstrate such an association.
There is no doubt, however, that future developments will render these biological markers
as useful diagnostic, prognostic, and predictive
tools. Array technologies have made the examination of genetic events possible and for thousands of genes to be monitored simultaneously,
making it possible to better understand the
events that characterize the different stages of
cancer development. Nevertheless, to achieve
useful clinical goals, we need well-designed translational research programs and well-conducted
prospective trials.
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