Introduction
Each year approximately 500,000 people worldwide are diagnosed with squamous cell carcinoma of the
head and neck (HNSCC). Advances in radiation therapy, chemotherapy and surgical techniques have
certainly improved quality of life, but have not been very successful in extending patient survival. One
major obstacle to curing patients is that tumor behavior is not easily predicted or explained by
traditional histopathologic methods. In fact, clinical outcomes and histologic findings are often
widely disparate. Such limitations have prompted a search for novel markers to diagnose, stage,
prognosticate, and treat HNSCC. A biomarker is a biochemical, molecular or
genetic parameter that can be objectively measured or evaluated to discern the presence and progress of
disease. With the onset of the molecular revolution, the armament of potential biomarkers has been
greatly expanded as the molecular pathways involved in tumor initiation and progression become
increasingly understood.
Potentially Useful Biomarkers in HNSCC
Cell Cycle Regulation
The p53 gene stands at the center of critical pathways controlling cell growth through the cell
cycle and response to stress through apoptosis. Alterations in the p53 gene are among the most common
tumor-related genetic events in HNSCC. Over 50% of HNSCCs harbor p53 gene mutations, and over 50% show
loss of the chromosome region 17p13 – the region where the p53 gene resides. P53 sequencing is
technically difficult, costly, and time-consuming. Opportunely, most mutations result in conformational
changes in tertiary folding resulting in stabilized protein that can be readily detected by
immunohistochemical staining.
The p16 gene is a key component of the Retinoblastoma (Rb) gene pathway. Its protein prohibits
cells from entering the cell cycle by inhibiting the cyclin dependent kinases 4 and 6. Inactivation of
p16 can occur by any combination of promoter hypermethylation, gene mutation and loss of heterozygosity
(LOH). Indeed, LOH at chromosomal region 9p21-22 (where p16 resides) is the most common of all genetic
alterations associated with HNSCC.
Cyclin D1 (also known as PRAD1) is a proto-oncogene that associates with cyclin-dependent
kinases 4 and 6 to phosphorylate Rb. High cyclin D1 expression (just like p16 inactivation) thus
facilitates progression from G1 to S phase. Cyclin D1 is located on chromosomal region 11q13,
and this region is amplified in approximately one-third of HNSCCs.
Signal Transduction
Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase. It is a key factor
in signal transduction of cellular response to a variety of growth-stimulating factors. It is
overexpressed in most HNSCCs and is amplified in about 10% of primary tumors. EGFR is an attractive
target of novel therapies.
One of the properties of malignancy is the production of angiogenic factors that induce new blood
vessel formation, facilitating further tumor growth through the provision of increased delivery of
nutrients. Vascular endothelial growth factor (VEGF) is induced under hypoxic conditions (e.g.
tumor hypoxia), and its expression induces proliferation, migration and survival of endothelial cells
during tumor growth by binding to specific tyrosine receptor kinases. Like EGFR, VEGF is an attractive
target of novel therapies.
Prostaglandin Metabolism
The cyclo-oxygenase 2 (Cox-2) enzyme catalyzes the rate-limiting step in the conversion of
arachodonic acid into prostaglandins and thromboxanes. Cox-2 contributes to carcinogenesis by catalyzing
the synthesis of mutagens, decreasing apoptosis, increasing inflammation and immunosuppression, and
enhancing the potential for invasion and metastasis. A number of studies suggest that Cox-2 activity is
important in the progression of epithelial cancers.
Extracellular Matrix Degradation
The matrix metalloproteinases MMPs are a family of zinc-dependent proteolytic enzymes that
degrade the basement membrane and other components of the extracellular matrix. Expression of MMPs by
HNSCCs may play an important role in tumor invasion and metastasis.
Oncoviruses
The 16 subtype of human papillomavirus (HPV-16) has recently been established as a common
causative agent for a majority of those HNSCCs arising from the oropharynx. Viral integration into the
host genome permits expression of the viral oncoproteins E6 and E7. E6 binds and degrades p53 protein
while E7 binds and degrades Rb protein, thus disrupting two critical pathways of tumorigenesis.
Potential Applications of Biomarkers for HNSCC
In the past, biomarker analysis has mainly focused on their role as prognostic indicators. More
recently, the role of biomarkers has been greatly expanded to address all aspects of patient care. In
the context of HNSCC, the diverse roles of biomarkers include:
Cancer Screening and Early Cancer Diagnosis
Identification of high risk populations
Identification of high risk premalignant lesions:
Although premalignant lesions (i.e. dysplasia) of the upper aerodigestive tract are at risk of
progressing to overtly malignant HNSCC, the measurement of histologic parameters to assess the likelihood
of progression is fraught with difficulties: The histopathologic features of premalignant lesions can be
subtle and often overlap with non-neoplastic reactive processes; and interpretation of these morphologic
features is subjective such that there is considerable variation among pathologists in the recognition
and grading of premalignant lesions.
Among the biomarkers that may help identify those precursor lesions that are most likely to develop
into HNSCCs, LOH at defined chromosomal loci is perhaps the most promising. Several studies on oral
dysplasias have shown that dual LOH on 3p and 9p can reliably distinguish those lesions that will
progress to HNSCC from those lesions that will not progress. Large scale genomic status (ploidy) has
also been highly touted as a means to identify high risk dysplastic lesions, with aneuploid status
associated with a high likelihood of progression to HNSCC. Recent concerns over the integrity of some of
these findings calls for more studies to assess the strength of aneuploid status as a marker of
progression.
Detection of undiagnosed HNSCC:
One of the more promising breakthroughs regarding early cancer diagnosis has been the ability to use
saliva as a substrate for biomarker assessment. Saliva has been used as a noninvasive, inexpensive and
readily accessible diagnostic substrate to assess diverse biomarkers including LOH status, HPV status,
promoter hypermethylation profile, p53 gene mutations, differential gene expression profiles, and
others. For patients with oral HNSCCs, the methylation profiles and HPV status of their tumors can be
discerned from molecular genetic analysis of their oral washes. Despite the documented feasibility of
saliva-based strategies, early detection saliva assays have failed to yield a high enough sensitivity and
specificity for broad population-based screening.
Tumor Staging
Tumor localization
A significant subset of patients with HNSCC present with metastatic spread to cervical lymph nodes in
the absence of a primary tumor by clinical, radiographic, endoscopic and even histopathologic
evaluation. Biomarkers can be used to help pinpoint primary site of tumor origin. One biomarker
approach takes advantage of the fact that some HNSCCs are caused by certain oncogenic viruses that target
specific regions of the upper aerodigestive tract. In effect, detection of a specific virus in the
metastasis implicates site of tumor origin. The potential role of viral probes as a tool to localize
tumor origin has recently been expanded with the recognition that HPV-16 is an important causative factor
in the development of 40-60% of HNSCCs arising from the oropharynx, but not for HNSCCs arising from
non-oropharyngeal sites. In-situ hybridization detection of HPV-16 in a metastatic implant reliable
points to the oropharynx as the site of tumor origin. Opportunely, p16 is overexpressed in tumors
associated with high-risk HPV to levels that are easily discerned by routine immunohistochemistry (IHC).
In effect, p16 in HNSCCs is a reliable surrogate marker of HPV infection such that p16 immunostaining of
a lymph node metastasis provides a simple way to discern origin from the oropharynx.
local extent (e.g. surgical margin analysis)
Tumor extent
Ongoing molecular genetic studies are providing further insights into the behavior of HNSCC including
an emerging picture that phenotypically normal but genetically altered cells may extend well beyond the
gross and microscopic confines of a tumor. These observations have introduced the intriguing concept of
biomarker assessment of surgical margins as a means of determining adequacy of resection. The potential
usefulness of biomarker analysis of surgical margins has since been confirmed in studies looking at p53
mutations, LOH, and eIF4E proto-oncogene overexpression.
Prognostication
The vast majority of biomarker studies have focused on their role as a prognostic factor – "an objective property from which the likely course of a
disease can be inferred". Despite a bewilderingly large body of studies evaluating the prognostic
significance of cell proliferation (e.g. Ki67, PCNA), p53 immunohistochemistry, aptosis, cytometry,
EGFR, bcl2 and many other biomarkers, few none have consistently proved reliable across multiple studies
and none are currently used in routine surgical pathology practice.
The modest prognostic impact of currently used biomarkers is not altogether surprising given the
complexity of HNSCC tumorigenesis requiring the concerted actions of multiple genes. Rapidly advancing
technology now permits comprehensive analysis of global gene expression during the progressive stages of
HNSCC tumorigenesis in ways that could identify specific expression "signatures" for tumor invasion,
metastasis and treatment responsiveness.
Treatment Selection
Radiation sensitive vs. radiation resistant; chemosensitive vs. chemoresistant
Selection for molecular targeted therapy:
Molecular-targeted therapies for HNSCC are rapidly becoming a reality. A number of phase III trials
in patients with advanced HNSCC are already underway. The most promising of these drugs target EGFR:
Erbitux (Cetuximab®), a monoclonal EFGR antibody, has shown efficacy in combination with radiotherapy in
a recent phase III trial; and erlotinib (Tarceva®), an EGFR tyrosine kinase
inhibitor, has shown favorable results in phase II trials as a monotherapy and in combination with
chemotherapy. Trial design now rests on the use of appropriate biomarkers to effectively identify those
patients who will benefit from EGFR-targeted therapy.
Confirmation of oncogenic HPV as an important causative agent in oropharyngeal cancer has opened the
door for vaccine immunotherapy. Recent studies showing the effectiveness of HPV vaccines in blocking the
development of cervical cancer raises hope that a similar approach can be more widely employing to
HPV-related HNSCCs.
Post-treatment Monitoring
Tumor DNA is shed into body fluids such as plasma and saliva. Accordingly, sensitive molecular
genetic methods to detect tumor specific alterations (e.g. microsatellite alterations, promoter
hypermethylation, HPV DNA) can be employed in these substrates to discern the presence and progression of
tumor independent of clinical, radiographic or histopathologic findings. Detection of circulating tumor
DNA, in turn, could potentially separate those patients who might benefit from intense chemotherapy and
those who can forego such toxic treatment.
References
- Begum S, Gillison ML, Ansari-Lari MA, et al. 2003 Clin Cancer Res 9:6469-75.
- Brennan JA, Mao L, Hruban RH, et al. 1995 N Engl J Med 16:429-35.
- Chai RL, Grandis JR. 2006 Curr Treat Options Oncol 7:3-11.
- Chung CH, Levy S, Yarbrough WG. 2005 Head Neck 28:360-8.
- Gillison ML, Koch WM, Capone RB, et al. 2000 J Natl.Cancer Inst 92:709-20.
- Kim MM, Califano JA. 2004 Int J Cancer 112:545-53.
- Koch W, Sidransky D. 2004 Semin Radiat Oncol 14:130-8.
- Nawroz-Danish H, Eisenberger CF, Yoo GH, et al. 2004 Int J Cancer 111:96-100.
- Rosin MP, Cheng X, Poh C, et al. 2000 Clin Cancer Res 6:357-62.
- Zhao M,.Rosenbaum E,.Carvalho A, et al. 2005 Int J Cancer 117:605-10.
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