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Journal of Clinical Oncology, Vol 25, No 11 (April 10), 2007: pp. 1369-1376
© 2007 American Society of Clinical Oncology.
DOI: 10.1200/JCO.2005.05.3397
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Identification of Angiogenesis/Metastases Genes Predicting Chemoradiotherapy Response in Patients With Laryngopharyngeal Carcinoma

Ian Ganly, Simon Talbot, Diane Carlson, Agnes Viale, Ellie Maghami, Iman Osman, Eric Sherman, David Pfister, Shaokun Chuai, Ashok R. Shaha, Dennis Kraus, Jatin P. Shah, Nicholas D. Socci, Bhuvanesh Singh

From the Laboratory of Epithelial Cancer Biology; Head and Neck Service, Department of Surgery Department of Pathology; Microarray Core Facility; Department of Medical Oncology; Department of Epidemiology and Biostatistics; and the Department of Computational Biology, Memorial Sloan-Kettering Cancer Center, New York, NY

Address reprint requests to Bhuvanesh Singh, MD, PhD, Laboratory of Epithelial Cancer Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021; e-mail: singhb{at}mskcc.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 GLOSSARY
 Appendix
 REFERENCES
 
Purpose To identify genes related to angiogenesis/metastasis that predict locoregional failure in patients with laryngopharyngeal cancer (LPC) undergoing chemoradiotherapy (CRT) treatment.

Methods Tumor tissue was collected and snap-frozen from 35 sequential patients with histologically confirmed LPC being treated with CRT. Gene expression analysis was performed using a novel cDNA array consisting of 277 genes functionally associated with angiogenesis (n = 152) and/or metastasis (n = 125). Locoregional response was correlated to the gene expression profiles to identify genes associated with outcome. These genes were internally validated by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and validated externally by immunohistochemistry analysis on an independent set of patients.

Results Locoregional failure occurred in nine of 35 patients. Seventeen genes from the cDNA microarray correlated with locoregional failure (two-sample t test, P < .05). Seven genes were chosen for additional analysis based on the availability of antibodies for immunohistochemistry. Of these seven genes, real-time RT-PCR validated four genes: MDM2, VCAM-1, erbB2, and H-ras (Wilcoxon rank sum test, P = .008, .02, .04, and .04, respectively). External validation by immunohistochemistry confirmed MDM2 and erbB2 as being predictive of locoregional response. Controlling for stage of disease, positivity for MDM2 or erbB2 was an independent negative predictor of locoregional disease-free survival.

Conclusion Genomic screening by cDNA microarray and validation internally by real-time RT-PCR and externally by immunohistochemistry have identified two genes (MDM2 and erbB2) as predictors of locoregional failure in LPC patients treated with CRT. The role of these genes in treatment selection and the functional basis for their activity in CRT response merit additional consideration.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 GLOSSARY
 Appendix
 REFERENCES
 
Organ-preservation chemoradiotherapy (CRT) is an acceptable alternative to conventional surgical treatment for laryngopharyngeal cancer (LPC). Several reports have shown that CRT provides comparable survival outcome for cancers of the larynx, hypopharynx, and oropharynx, with preservation of the larynx in 60% to 80% of patients with laryngeal and hypopharyngeal cancer.1-5 However, CRT is not effective in all patients. When unsuccessful, patients suffer unnecessarily from treatment toxicity and require complication-prone salvage surgery.6 Reliable predictors of outcome are needed to identify patients whose tumors are most suitable for CRT versus those best treated with primary surgery.

Molecular markers may have a significant role to play in the identification of such patients. For example, some studies have reported that genes controlling the apoptosis pathway, such as p53 and the Bcl-2 family of proteins, may help distinguish tumors sensitive to CRT.7-10 Identification of novel molecular markers can now be facilitated by microarray analysis, allowing for simultaneous screening of multiple candidates. Such technology has been reported to identify genes predictive of response to chemotherapy in many tumor types,11-15 although not yet in head and neck cancer. We focused on the molecular pathways involved in angiogenesis and metastases, given that studies suggest they are key factors in determining treatment responsiveness.16-18 Using a cDNA microarray constructed in our laboratory, we were able to identify 17 genes that were associated significantly with locoregional failure. These genes were subjected to internal validation using real-time reverse transcriptase polymerase chain reaction (RT-PCR) followed by external validation by IHC on an independent set of patients from a clinical trials data set. Two genes (MDM2 and erbB2) remained predictors of locoregional tumor response to CRT after the validation analyses.


   METHODS
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 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 GLOSSARY
 Appendix
 REFERENCES
 
Sample Collection and Pathologic Review
After informed consent was obtained, the guidelines of the institutional review board were followed and tumor tissue for the test group was collected and snap-frozen from 35 sequential patients with histologically confirmed LPC before definitive treatment with CRT. Details on patients included in the validation group have been published previously.9 In brief, these patients were part of two separate but related CRT clinical trials consisting of 62 patients with LPC.9 Details of chemotherapeutic management were published previously.4,5,9

Preparation of cDNA Microarrays
Gene expression analysis was performed on all samples using a cDNA array consisting of 277 genes functionally associated with angiogenesis (n = 152) and/or metastasis (n = 125) identified by a comprehensive review of the literature. Sequence-verified human IMAGE cDNA clones were PCR amplified, and spotted in high-density 20 replicates onto polylysine-coated microscope slides using a custom robot. A total of 1,152 randomly selected genes were also spotted onto the array for the purpose of intersample normalization. Before hybridization, slides were handled as described previously.19

Labeling of cDNA and Hybridization to Arrays
RNA was extracted from freshly frozen pretreatment biopsies through a Trizol extraction followed by isolation using RNeasy columns (Qiagen, Valencia, CA). Ten micrograms of total RNA from each tumor sample was used to synthesize cDNA with 2 U of reverse transcriptase (Fairplay Kit; Stratagene, La Jolla, CA). Universal normal RNA (Stratagene) was used as the reference RNA. Tumor cDNA was labeled with cyanine 5–conjugated deoxyuridine 5'-triphosphate (Amersham Biosciences, Piscataway, NJ) and universal normal labeled with cyanine 3–conjugated deoxyuridine 5'-triphosphate (Amersham Biosciences). Hybridization to cDNA arrays was carried out overnight at 50°C in a buffer containing 30% formamide, 3x saline sodium citrate (SSC), 0.75% sodium dodecyl sulfate, and 100 ng of human Cot-1 DNA (Invitrogen, Carlsbad, CA). After hybridization, slides were washed in 0.2x SSC/0.1% sodium dodecyl sulfate at room temperature for 1 minute and in 0.2x SSC at room temperature for 15 minutes. Slides were dried and then scanned using a GenePix Pro 4000A microarray scanner (Axon Instruments, Foster City, CA).

Collection and Statistical Analysis of Microarray Data
Red (Cy5) and green (Cy3) fluorescent signal intensities for each spot on the array were calculated using the GenePix Pro 3.0 software (Axon Instruments). The data were normalized using an intensity-dependent nonlinear normalization scheme described previously.19-22 Only genes that had an absolute log-fold change of log2(3) (ie, log base 2) in at least four of the samples (25%) were retained for analyses. These analyses included both a comparison of global gene expression profiles using clustering methodology and a comparison of expression of individual genes among the samples. Tumor samples were clustered using the hierarchical Ward linkage method and then significance was assessed by nonparametric bootstrap resampling.23,24 We then applied a nonparametric two-sample test to find individual genes for which expression differentiated locoregional response. We computed the P value for each gene using the Wilcoxon rank sum test. Significance was accepted at a two-tailed P < .05.

Quantitative Real-Time PCR Copy Number Analysis
Expression changes in the genes identified by the cDNA microarray were validated and quantified using real-time RT-PCR with the ICycler System (Bio-Rad Laboratories, Hercules, CA) using SYBR-green detection. PCR amplification of an 80- to 150-bp fragment of each gene was performed using forward and reverse primers. Forward and reverse primers, product size, and optimum annealing temperature for each gene are listed in Table 1.


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  		Table 1. Forward and Reverse Primers, Product Size, and Optimum Annealing Temperature for Each Gene

 
As a reference gene, the housekeeping gene 18S rRNA was amplified using the following primers: forward 5'-GTAACCCGTTGAACCCCATT and reverse 5'-CCATCCAATCGGTAGTAGCG25 using an annealing temperature of 55°C. The ICycler real-time PCR reaction was performed in a 25-µL reaction containing the SYBR Green PCR Master Mix (PE Applied Biosciences, Foster City, CA), 20 nmol/L primers, and DNA template solution (2 µL). The following ICycler run protocol was used: denaturation program (95°C for 10 minutes), amplification, and quantification program repeated for 35 to 40 cycles (95°C for 30 seconds, 55 to 60°C for 30 seconds, 72°C for 30 seconds). Amplification was followed by melting curve analysis to ensure the presence of a single PCR product.26 After computation of the reaction efficiencies from the standard curve constructed from serial dilutions of normal universal cDNA, copy number ratios between the gene of interest and 18S were calculated.

IHC
Five-micrometer sections from each paraffin-embedded tumor were cut and placed on polylysine-coated slides. Representative sections were stained with hematoxylin and eosin and analyzed to confirm the presence of the desired target tissue. Corresponding sections were subjected to immunohistochemical analysis. Sections were deparaffinized, rehydrated in graded alcohols, and processed using the avidin-biotin immunoperoxidase method. Immunostaining was performed using the Ventana automated stainer according to the manufacturer's recommendations (Ventana Medical Systems, Tucson, AZ). Diaminobenzidine was used as the chromogen and hematoxylin was used as the nuclear counterstain. The following antibodies were used: H-ras (Santa Cruz sc-29 mouse monoclonal at a dilution of 1/250; Santa Cruz Biotechnology, Santa Cruz, CA), VCAM-1 (Santa Cruz sc-13160 mouse monoclonal at a dilution of 1/1000), erbB2 (DAKO A0485 rabbit polyclonal; DAKO, Carpinteria, CA), and MDM2 (mouse monoclonal 2A10 at a dilution of 1/500). Tissue known to express each protein was used as positive controls, colon tissue was used for MDM2 and H-ras, and breast tissue for erbB2 and tonsil tissue for VCAM-1. The tissue was scored by an independent pathologist blinded to the study. Samples were given a histoscore of 0, 1, 2, or 3 according to the intensity of staining and the percentage of cells staining positively. Tissues with a histoscore of 0 and 1 were coded as negative and those with a histoscore of 2 or 3 were coded as positive.

Statistical Analysis of Clinical Outcomes
Clinical and pathologic data for the training set (cDNA microarray patients) and for the validation set (IHC patients) were entered into a computer database (Microsoft Excel 2000; Microsoft Corp, Redmond, WA). The follow-up interval was calculated in months from the date of first treatment to the date of the last follow-up or death, and locoregional disease-free interval was calculated from the date of first treatment to the date of first local or regional recurrence. The training set had a median follow-up time of 21 months (range, 12 to 45 months). Nine of 35 patients had locoregional recurrence within 12 months of index therapy (mean time to locoregional recurrence, 5.3 months). Training set patients were coded as responders or nonresponders and a binary analysis was performed using a t test to compare gene expression from the cDNA microarray. Similarly, a binary analysis was used to compare the real-time PCR results between responders and nonresponders. The validation set survival analysis was carried out using the Kaplan-Meier method. Locoregional disease-free survival rates were calculated for each protein (MDM2, erbB2, VCAM-1, H-ras) using a commercially available computer software package (JMP 4.0; SAS Institute Inc, Cary, NC). Univariate comparisons were then performed using the log-rank test. Controlling for stage of disease, multivariate analysis was performed using a Cox proportional hazards model.


   RESULTS
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 GLOSSARY
 Appendix
 REFERENCES
 
Identification of Angiogenesis/Metastases Genes by Microarray Analysis
Locoregional failure occurred in nine of 35 patients. Global gene expression of responders and nonresponders was assessed using cDNA microarray analysis. No stable hierarchical clustering was identified after bootstrap resampling of unsupervised data. On direct analysis, we identified 17 of 277 genes that correlated with locoregional response (P < .05). The gene symbols, Entrez gene ID number, gene description, level of expression, and P values are listed in Table 2. When responders were compared with nonresponders, six genes were overexpressed and 11 genes had loss of expression. Seven genes (shown in bold type in Table 2) were chosen for further analysis based on clinical and biologic relevance and availability of antibodies for IHC. These genes were H-ras, NME4, CDH13, VCAM-1, EGFR, MDM2, and erbB2.


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  		Table 2. Genes Predictive of Locoregional Response Validated by Real-Time Reverse Transcriptase Polymerase Chain Reaction

 
Internal Validation of Predictive Genes by Quantitative Real-Time PCR
Real-time PCR was used to quantify the expression of mRNA in these seven genes. Expression levels were then correlated to locoregional response. Four of the seven genes were validated by real-time PCR (shown in bold type in Table 3): MDM2, VCAM-1, erbB2, and H-ras (Wilcoxon rank sum test, P = .008, .024, .041, and .035, respectively).


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  		Table 3. Genes Predictive of Locoregional Response Validated by Real-Time Polymerase Chain Reaction

 
External Validation of Predictive Genes by IHC of Protein Expression
On the basis of a cDNA array of 277 genes and P < .05, we would expect 14 genes to occur by chance. Therefore, it was important to validate both the cDNA array results and real-time PCR results on an external validation cohort. We chose an independent set of tumors from 62 patients with LPC treated by chemoradiotherapy as described previously and carried out IHC for MDM2, erbB2, VCAM-1, and H-ras. Tumors with a histoscore of 0 or 1 were coded as negative and those with a histoscore of 2 or 3 were coded as positive (Fig 1). Results were then correlated to locoregional response and are listed in Table 4. MDM2 was dysregulated in 66.1% of samples. The Kaplan and Meier plots for MDM2 status are shown in Figure 2; MDM2 positivity correlated with poorer locoregional disease-free survival by the log-rank test (P = .034). Controlling for stage of disease, multivariate analysis showed that MDM2 positivity was an independent predictor for poorer locoregional disease-free survival (Table 5, part A). erbB2 was dysregulated in 22.6% of samples. Although erbB2 positivity correlated with poorer locoregional disease-free survival, this was not statistically significant (P = .12; Fig 3). In addition, multivariate analysis showed that erbB2 positivity was not an independent predictor (Table 5, part B). Both VCAM-1 and H-ras were not validated by IHC. Comparing MDM2-negativity/erbB2-negativity status to positivity for either MDM2 or erbB2 showed widening in the locoregional survival curves (P = .008; Fig 4). Controlling for stage of disease, multivariate analysis showed that MDM2 or erbB2 positivity was associated with an increased risk of locoregional failure, with a hazard ratio of 2.55 (P = .008; Table 5, part C).


Figure 1
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  		Fig 1. Immunohistochemical analysis of erbB2, VCAM, H-ras, and MDM2 in head and neck squamous cell carcinoma tumors. Histoscores of 0 and 1 are coded as negative, and those with 2 or 3 are coded as positive.

 

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  		Table 4. Immunohistochemical Analysis Showing Percentage of Samples Dysregulated for Each Protein and Correlation With LR-DFS

 

Figure 2
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  		Fig 2. Kaplan-Meier curves for locoregional disease-free survival stratified by MDM2 expression. Curves are compared using log-rank test.

 

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  		Table 5. Multivariate Analysis Controlling for Stage of Disease

 

Figure 3
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  		Fig 3. Kaplan-Meier curves for locoregional disease-free survival stratified by erbB2 expression. Curves are compared using log-rank test.

 

Figure 4
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  		Fig 4. Kaplan-Meier curves for locoregional disease-free survival stratified by MDM2 and erbB2 expression. Curves are compared using log-rank test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 GLOSSARY
 Appendix
 REFERENCES
 
Organ-preserving CRT is an emerging alternative treatment for locally advanced cancers of the larynx,1,27,28 hypopharynx,2 and oropharynx3,9; it allows preservation of speech and swallowing functions that would be affected adversely by surgical resection. However, not all patients respond to this therapy; in laryngeal cancer 20% to 40% of patients will require a salvage total laryngectomy. Identification of patients who are not likely to respond to CRT would avoid the unnecessary toxicity associated with this treatment and allow patients to be treated more appropriately by primary surgical resection.

At present there are no clear markers, either clinical or molecular, that can predict CRT response reliably in these patients. Reports on individual genes, such as p537-9,29,30 and the Bcl-2 family of proteins,10,31 have produced conflicting results. Some of these conflicting observations with both p53 and the Bcl-2 family of proteins may be explained by the fact these studies reported on different tumor subsites of the head and neck (ie, oropharynx, hypopharynx, and larynx). This could suggest that expression of these markers may be site specific. However, this observation is better explained by the fact that there are many other genes involved in the CRT response, which include enzymes involved in apoptosis, DNA repair and metabolism, and detoxification of drugs. Expression of these proteins will vary with individual response. Hence distinguishing responders from nonresponders before treatment is started requires analysis of a larger set of genes to act as predictive markers.

In this study, our patient population comprised 13 laryngeal, four hypopharyngeal, and 18 oropharyngeal cancers. We chose this group of patients to identify markers predictive of CRT response applicable to a variety of tumors. We used microarray technology, which has been established as an efficacious approach for the identification of prognostic markers.32 The use of microarrays to generate expression profiles associated with sensitivity of cancer cells to drugs has been reported by Scherf et al.33 Several others have reported on specific tumor types.11-15 The use of such technology not only helps identify patients most likely to respond to treatment, but also allows us to better understand the molecular pathways involved in response, thus allowing us to better identify new targets for drugs and design new treatment strategies for patients.

Although microarray technology has been used successfully for chemoresponse assessment in many other cancers, there is little in the literature on head and neck squamous cell carcinoma. We were particularly interested in genes involved in angiogenesis and metastases because recent reports had shown that these factors are important in the CRT response.16-18 From an array of 277 genes, we identified 17 genes that correlated with response. These 17 genes comprised several genes not previously believed to be important in head and neck cancer, although powerful, array-based identification of prognostic markers is fraught with false-positive results. Accordingly, both internal and external validation is needed to confirm results from array screening. In our study, we internally validated the findings of cDNA array analysis by real-time PCR and externally validated our findings on an independent cohort of tumors by IHC. Of seven genes chosen for additional analysis, we were able to validate MDM2 as being significant, with a trend to significance for erbB2.

Our finding that MDM2 overexpression is predictive of poorer CRT response is supported by evidence in the literature. For example, it has been reported that in vitro, overexpression of MDM2 increases growth rate,34 and transgenic mice overexpressing MDM2 have an increased incidence of lymphoma and sarcoma formation.35 Ikeguchi et al36 has reported that MDM2 overexpression correlated with a lack of CRT response in esophageal squamous cell carcinoma patients. In addition, antisense oligonucleotides targeted against MDM2 have been reported recently as therapies in tumors overexpressing MDM2.37-40 Both Bianco et al39 and Wang et al40 also have shown that antisense oligonucleotides to MDM2 enhance the chemotherapeutic efficacy of a wide range of cytotoxic agents by p53-dependent and p53-independent mechanisms. Clearly, MDM2 is important in carcinogenesis and in chemoradiosensitivity. This finding therefore suggests additional investigation of MDM2 as a predictor of response to CRT in laryngopharyngeal cancer is warranted.

Our study also suggests erbB2 overexpression is predictive of poorer CRT response. erbB2 (Her-2/neu) is a member of the tyrosine kinase superfamily,41 and overexpression leads to increased basal tyrosine kinase activity, which transforms the cell by chronically stimulating signal transduction pathways. Overexpression has been shown in many tumor types, including breast42 and prostate.43 Studies of erbB2 in head and neck squamous cell carcinoma are few and results are contradictory. The reported incidence of overexpression ranges from 0% to 47%, with conflicting correlations with clinical outcome.44-51 Such variations in overexpression and correlation to outcome may be due to differences in IHC techniques used, such as different antibodies, antigen retrieval methods, and pathology reporting. IHC for erbB2 is used routinely at our institution for the evaluation of breast cancer specimens. By using the same method of IHC and a pathologist blinded to the study, we were confident that the results we obtained were reflective of erbB2 expression in the tumors studied. We were able to show that overexpression of erbB2 occurred in 23% of head and neck squamous cell carcinoma patients studied. We were also able to correlate this to response, showing a trend toward significance. An association between erbB2 positivity and chemosensitivity or radiosensitivity has already been established in other tumor types. For example, Akamatsu et al52 reported that overexpression of erbB2 in esophageal cancer was related to chemoradioresistance, and Nishioka et al53 reported radioresistance in cervical cancer correlated with overexpression of erbB2. In head and neck cancer, Uno et al54 reported that recombinant humanized anti-erbB2 monoclonal antibody (rhuMab HER-2) in combination with radiation had additive efficacy in head and neck cancer cells. These studies, together with our own findings, suggest erbB2 has role in CRT response in head and neck cancer. When both molecular factors are combined, we have shown that MDM2 or erbB2 positivity by IHC correlates with a poorer locoregional disease-free survival as well as overall survival. We therefore propose that the combination of these two markers may prove to be a useful predictor of CRT response.

In conclusion, we believe that genomic screening and real-time PCR analyses have identified and validated four genes predictive for locoregional response in LPC patients treated with CRT. IHC on an independent cohort of tumors validated one of these genes (MDM2) and showed a trend toward significance for erbB2. A positive IHC for either MDM2 or erbB2 is an independent predictor of locoregional disease-free survival by multivariate analysis. Additional analysis of MDM2 and erbB2 on a larger cohort of patients is warranted.


   AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 AUTHORS' DISCLOSURES OF...
AUTHOR CONTRIBUTIONS
 GLOSSARY
 Appendix
 REFERENCES
 
The authors indicated no potential conflicts of interest.


   AUTHOR CONTRIBUTIONS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
GLOSSARY
 Appendix
 REFERENCES
 
Conception and design: Ian Ganly, Ellie Maghami, David Pfister, Bhuvanesh Singh

Financial support: Bhuvanesh Singh

Administrative support: Jatin P. Shah, Bhuvanesh Singh

Provision of study materials or patients: Simon Talbot, Agnes Viale, Eric Sherman, David Pfister, Ashok R. Shaha, Dennis Kraus, Jatin P. Shah, Bhuvanesh Singh

Collection and assembly of data: Ian Ganly, Simon Talbot, Agnes Viale, Ellie Maghami, Eric Sherman, David Pfister, Shaokun Chuai, Bhuvanesh Singh

Data analysis and interpretation: Ian Ganly, Diane Carlson, Ellie Maghami, Iman Osman, David Pfister, Nicholas D. Socci, Bhuvanesh Singh

Manuscript writing: Ian Ganly, Bhuvanesh Singh

Final approval of manuscript: Ian Ganly, Simon Talbot, Ellie Maghami, David Pfister, Ashok R. Shaha, Dennis Kraus, Jatin P. Shah, Bhuvanesh Singh

Other: Diane Carlson, Ellie Maghami


   GLOSSARY
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 GLOSSARY
Appendix
 REFERENCES
 

erbB2:
Also called the human epithelial growth factor receptor (HER), erbB belongs to the EGFR receptor family. erbB1 (EGFR/HER-1), erbB2 (HER-2), erbB3 (HER-3), and erbB4 (HER-4) are the four members that comprise this receptor family.

H-ras:
Isoform of Ras.

Locoregional failure:
Failure at the primary site or the regional lymphatics.

MDM2:
An E3 ubiquitin ligase that recognizes the N-terminal activation domain (TAD) of proteins belonging to the p53 family. By blocking the ability of p53 to associate with factors involved in protein transcription, the MDM2 interaction with p53 prevents transcriptional activation. Furthermore, interaction with p53 leads to ubiquitinylation and subsequent degradation of p53.

VCAM-1 (vascular cell adhesion molecule):
A member of the superfamily of immunoglobulin-like molecules, VCAM is expressed on endothelial cells and regulates leukocyte migration across blood vessels. The molecule is a transmembrane protein that binds to a cytoskeletal protein within the cell and to the same (homophilic) or a different (heterophilic) protein present on another cell. It is also an anchor for the developing vasculature during angiogenesis.


   Appendix
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 ABSTRACT
 INTRODUCTION
 METHODS
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 DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 GLOSSARY
 Appendix
REFERENCES
 
Go


Figure 5
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  		Fig A1. Dentogram showing clusters of gene similarities between tumor samples.

 


   NOTES
 
Presented at the Annual Meeting of the American Association for Cancer Research, March 27-31, 2004, Orlando, FL.

Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 AUTHORS' DISCLOSURES OF...
 AUTHOR CONTRIBUTIONS
 GLOSSARY
 Appendix
 REFERENCES
 
1. Wolf G, Hong K, Fisher S, et al: Induction chemotherapy plus radiation compared with surgery plus radiation in patients with advanced laryngeal cancer: The Department of Veterans Affairs Laryngeal Cancer Study Group. N Engl J Med 324:1685-1690, 1991[Abstract]

2. Lefebvre JL, Chevalier D, Luboinski B, et al: Larynx preservation in pyriform sinus cancer: Preliminary results of a European Organization for Research and Treatment of Cancer phase III trial. J Natl Cancer Inst 34:890-899, 1996

3. Giralt JL, Gonzalez J, del Campo, JM, et al: Preoperative induction chemotherapy followed by concurrent chemoradiotherapy in advanced carcinoma of the oral cavity and oropharynx. Cancer 89:939-945, 2000[CrossRef][Medline]

4. Harrison LB, Pfister DG, Fass DE, et al: Concomitant chemotherapy-radiation therapy followed by hyperfractionated radiation therapy for advanced unresectable head and neck cancer. Int J Radiat Oncol Biol Phys 21:703-708, 1991[Medline]

5. Pfister DG, Harrison LB, Strong EW, et al: Organ function preservation in advanced oropharynx cancer: Results with induction chemotherapy and radiation. J Clin Oncol 13:671-680, 1995[Abstract/Free Full Text]

6. Ganly I, Patel SG, Matsuo J, et al: Postoperative complications of salvage total laryngectomy. Cancer 103:2073-2081, 2005[CrossRef][Medline]

7. Bradford CR, Zhu S, Wolf GT, et al: Overexpression of p53 predicts organ preservation using induction chemotherapy and radiation in patients with advanced laryngeal cancer: Department of Veterans Affairs Laryngeal Cancer Study Group. Otolaryngol Head Neck Surg 113:408-412, 1995[CrossRef][Medline]

8. Lavertu P, Adelstein DJ, Myles J, et al: p53 and Ki-67 as outcome predictors for advanced squamous cell cancers of the head and neck treated with chemoradiotherapy. Laryngoscope 111:1878-1891, 2001[CrossRef][Medline]

9. Osman I, Sherman E, Singh B, et al: Alteration of p53 pathway in squamous cell carcinoma of the head and neck: Impact on treatment outcome in patients treated with larynx preservation intent. J Clin Oncol 20:2980-2987, 2002[Abstract/Free Full Text]

10. Trask DK, Wolf GT, Bradford CR, et al: Expression of Bcl- family proteins in advanced laryngeal squamous cell carcinoma: Correlation with response to chemotherapy and organ preservation. Laryngoscope 112:638-644, 2002[CrossRef][Medline]

11. Kihari C, Tsunoda T, Tanaka T, et al: Prediction of sensitivity of esophageal tumors to adjuvant chemotherapy by cDNA microarray analysis of gene expression profiles. Cancer Res 61:6474-6479, 2001[Abstract/Free Full Text]

12. Okutsu J, Tsunoda T, Kaneta Y, et al: Prediction of chemosensitivity for patients with acute myeloid leukemia, according to expression levels of 28 genes selected by genome-wide complementary DNA microarray analysis. Mol Cancer Ther 1:1035-1042, 2002[Abstract/Free Full Text]

13. Mariadason JM, Arango D, Shi Q, et al: Gene expression profiling-based prediction of response of colon carcinoma cells to 5-fluorouracil and camptothecin. Cancer Res 15:8791-8812, 2003

14. Kikuchi T, Daigo Y, Katagiri T, et al: Expression profiles of non-small cell lung cancers on cDNA microarrays: Identification of genes for prediction of lymph-node metastasis and sensitivity to anticancer drugs. Oncogene 22:2192-2205, 2003[CrossRef][Medline]

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Submitted April 7, 2006; accepted January 5, 2007.


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