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Objective: To examine the role of MAGEA2 in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC)., Design: Primary tissue microarray data and quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed that MAGEA2 is differentially overexpressed in HNSCC. Functional analyses were then performed using MAGEA2 transfections and small-interfering RNA knockdowns with subsequent anchorage-dependent growth studies and cell cycle analyses. Quantitative RT-PCR was used to evaluate expression changes in p53 downstream targets after transfection of MAGEA2 into normal upper aerodigestive cell lines., Results: MAGEA2 is differentially overexpressed in HNSCC. In addition, MAGEA2 promotes growth in normal oral keratinocytes, whereas knockdown of MAGEA2 in HNSCC cells decreases growth. Using the HCT116 p53 wt and null cell line system, transfection of MAGEA2 induced growth in the p53 wt cell line while providing no growth advantage in the p53 mutant cells. Subsequently, transfection of MAGEA2 induced a decrease in messenger RNA expression of the p53 downstream targets CDKN1A and BAX and decreased G1 arrest in cells allowed to remain confluent for longer than 48 hours., Conclusions: These data suggest that MAGEA2 is differentially expressed in HNSCC and functions, in part, through the p53 pathway by increasing cellular proliferation and abrogating cell cycle arrest. This improved understanding of MAGEA2 function and expression patterns will potentially allow for the improved ability to use MAGEA2 for detection, surveillance, and targeted therapeutics.

Objective: To investigate if loss of extracellular matrix metalloprotease inducer (EMMPRIN) will inhibit the growth of head and neck squamous cell carcinoma (HNSCC) tumor cell lines in vivo. Tumor cell-derived EMMPRIN is highly overexpressed in HNSCC and is thought to be induced by surrounding fibroblasts to stimulate matrix metalloproteases, which modulate tumor cell invasion, growth, and angiogenesis., Design: In vivo study using FaDu tumor xenografts., Setting: Academic research facility., Subjects: Severe combined immunodeficiency (SCID) mice., Interventions: The HNSCC cell line FaDu was transfected with EMMPRIN (FaDu/E), control vector (FaDu), or plasmid-expressing small-interfering RNA against EMMPRIN (FaDu/siE). Tumor cells combined with fibroblast cells were xenografted onto the flank of SCID mice. Tumors were measured biweekly over 4 weeks, at which time the mice were killed, and tumor samples were analyzed for proliferation (Ki-67 immunohistochemical analysis), vascularization (factor VIII staining), and apoptosis (TUNEL [terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling] assay)., Main Outcome Measure: Growth of head and neck cancer cell lines genetically engineered to express variable levels of EMMPRIN., Results: Tumor growth positively correlated and animal survival negatively correlated with increasing EMMPRIN expression. FaDu/E tumor growth was significantly larger at 4 weeks compared with FaDu tumors (P = .006). Similarly, the control vector-transfected FaDu tumors were significantly larger than FaDu/siE (P < .001). Immunohistochemical analysis demonstrated increased Ki-67 in EMMPRIN-transfected cells, without a significant change in the rate of apoptosis between groups. Vascular density and tumor formation rate also increased significantly with EMMPRIN expression., Conclusion: This study suggests that anti-EMMPRIN-targeted therapy may prove to be a novel treatment option in HNSCC.

Objectives: To examine (1) the molecular angiogenic relationship between endothelial and stromal cells of angiofibromas and how this may elucidate the pathogenesis of angiofibromas and (2) the effects of embolization on the expression of angiotrophic factors and proapoptotic and antiapoptotic factors within the tumor., Design: The expression of mesenchymal and endothelial stem/progenitor cell-associated proteins (MECAPs) such as proangiogenic cytokine vascular endothelial growth factor (VEGF), VEGF receptors (VEGFR1, VEGFR2, and VEGFR3), angiopoietin receptors (Tie-1 and Tie-2), and stem cell subset marker CD133 was assessed by immunohistological staining in 7 embolized angiofibroma specimens. Expression of proapoptotic Bax, antiapoptotic Bcl-2 and Bcl-xL, nuclear proliferation protein MiB-1, and hypoxia-inducible factor 1alpha (Hif-1alpha) in peri-ischemic areas of the embolized angiofibromas was also assessed., Setting: A single pediatric institution., Patients: Seven patients (identified from medical records, January 1, 2001, through December 31, 2005) who were diagnosed as having juvenile angiofibroma and who underwent surgical treatment. Archival tissues were retrieved for immunostaining., Main Outcome Measures: The immunostaining results were evaluated by microscopy and the staining intensities were also recorded., Results: All angiofibroma specimens expressed the stem cell subset marker CD133 and MECAPs except VEGFR3 (a few cases). In the only case tested, we found evidence of VEGF-induced angiogenic signaling as the expression of phosphorylated VEGFR2 (Tyr951). Endothelial cells expressed VEGFR1 and VEGFR2 and angiopoietin receptors Tie-1 and Tie-2 but not VEGF. In contrast, VEGF was expressed within stromal cells. Viable tumor adjacent to the ischemic areas demonstrated increased staining intensities to VEGFR2, Tie-1, Tie-2 (all cases), and VEGFR3 (2 cases) and increased nuclear proliferation (5%-20%). All cases expressed proapoptotic and antiapoptotic factors, and the expression of Hif-1alpha was unaffected by ischemia., Conclusions: Stromal cells appear to be similar to mesenchymal stem cells with endothelial differentiation potential in umbilical cord blood cells. Stromal cells support endothelial growth by providing VEGF as a paracrine factor. Under ischemic stress, the embolized tumor tissues show upregulation of angiogenic receptors, retention of Hif-1alpha, and increased nuclear proliferation rates. Specific angiogenesis blockers may represent a novel treatment strategy for angiofibromas.

Objective: To investigate the prognostic significance of chromosomal aberrations of chromosomes X and 11 in relation to disease-specific survival in head and neck squamous cell carcinoma., Setting: University hospital., Design: A 10-year retrospective clinical study. Information about clinical findings, treatment, and follow-up has been recorded prospectively., Patients: By means of the fluorescence in situ hybridization technique with centromeric probes for chromosomes X and 11, we analyzed 40 randomly selected patients before treatment for T1 to T4 head and neck squamous cell carcinoma. Numerical aberrations were scored and evaluated in frozen sections., Main Outcome Measures: The significance of prognostic parameters was tested by the log-rank and Kaplan-Meier methods for the univariate analysis. The Cox proportional hazards regression model was used for multivariate analysis., Results: Numerical aberrations of chromosome 11 correlated positively with T and N classification (P = .03 and P = .02, respectively) and with clinical stage (P = .02). Patients with higher frequencies of numerical aberrations for both chromosome X (>48%, mean) and chromosome 11 (>57%, mean) had shortened disease-specific survival compared with those with lower frequencies of numerical aberrations (P = .008 and P<.001, respectively). Of patients who died from disease within 3 years, 7 (50%) had a trisomic value of chromosome 11 of 35% or higher of nuclei (P<.001). Moreover, patients with a higher value (>or=8%) of amplification of chromosome 11 (>4 signals) were associated with having poor prognosis compared with those with a lower value (P = .02)., Conclusion: Numerical aberrations of chromosomes X and 11 had prognostic value in head and neck squamous cell carcinoma, and higher frequencies of numerical aberrations correlated with poor prognosis.

Objective: To evaluate multiple genetic loci in patients with head and neck cancer to determine if, as in colorectal carcinoma, there is an orderly occurrence of genetic alterations, and if an accumulation of alterations affects patient survival., Design: Cohort study of patients with head and neck cancer in which fresh tissue was retrieved., Setting: Academic medical center., Patients: Forty-three patients treated surgically for squamous cell carcinoma of the head and neck from 1991 to 1994., Main Outcome Measures: The DNA from tumor and healthy tissue was evaluated for loss of heterozygosity at p53, retinoblastoma, and chromosome 16q and for amplification of cyclin D1. The respective RNA was probed for levels of p53, p 16, p21, and p27 messenger RNA. These findings were compared with tumor stage and patient survival., Results: DNA analysis showed that loss of heterozygosity occurred at p53 in 21% of tumors, at retinoblastoma in 35%, and at 16q in 21%, and that cyclin D1 was amplified in 42%. Messenger RNA levels of the assessed proteins were variably increased and decreased compared with healthy tissues obtained from the same patients with no discernable pattern. There was no correlation between any one of these genetic alterations and overall survival. When patients were analyzed for loss of heterozygosity at p53, retinoblastoma, 16q, or altered cyclin D1 in combination, 19 patients had no detectable alterations, 13 had 1, 6 had 2, and 5 had 3. Single genetic alterations did not affect survival; however, there was a trend toward decreased survival with multiple alterations. The 2-year Kaplan-Meier survival in patients with less than 1 genetic loss was 78% vs 58% in patients with 2 or more losses., Conclusions: The lack of a pattern of genetic alterations in head and neck cancer demonstrates that its progression can be mediated by a multitude of pathways, complicating its genetic evaluation. Single genetic alterations do not appear to affect survival; however, when multiple alterations are detected-regardless of combination-survival is affected. This observation lends credence to the theory that multiple genetic alterations contribute to cancer progression; however, the lack of a pattern of this genetic change is a significant obstacle to applying genetic findings to routine cancer therapy.

Objectives: To review recent advances in our understanding of programmed cell death, or apoptosis, and discuss implications of these basic science advances in our understanding of causes and potential treatments of a variety of diseases of the head and neck., Data Sources: Basic science literature relevant to the study of apoptosis and its clinical implications., Conclusions: Apoptosis is now understood to be important in the normal development and survival of all multicellular organisms. Deregulation of this normally tightly controlled process underlies a variety of disease states, including neoplasia, autoimmune disease, and disorders of the central nervous system. A better understanding of this process and its regulation may help otolaryngologists better understand diseases relevant to this specialty and will lead to improved therapeutic interventions.

Objective: To analyze and compare components of the 3 primary DNA repair pathways of Epstein-Barr virus-transformed lymphocyte (lymphoblastoid) cell lines derived from 9 patients with squamous cell carcinoma of the head and neck and 11 cancer-free controls. These cell lines were previously characterized by using an established cytogenetic marker of cancer susceptibility (mutagen sensitivity assay)., Design: To evaluate nucleotide excision repair (NER), we measured the reactivation level of a tobacco carcinogen-damaged plasmid containing a bacterial reporter gene transfected into these cells. To assess mismatch repair (MMR) and recombinational repair, selected gene transcript levels were quantified by using a multiplex reverse transcriptase-polymerase chain reaction assay. The results of these DNA repair assays were correlated with the previously measured mutagen sensitivity values., Results: The NER capacities of the 2 groups were similar: 25.1% (range, 14.3%-33.3%) for the patient cell lines and 26.0% (range, 9.4%-47.7%) for the control lines. Transcriptase levels for 6 MMR genes (hMSH3, hMSH2, hPMS2, GTBP, hMLH1 and hPMS1) did not differ in the 2 groups. Transcript levels for 4 of 6 recombinational repair genes (XRCC7, XRCC6, XRCC1, and RAD51) were higher in the patient cell lines, though this difference was significant only for XRCC7 (P = .003). The mutagen sensitivity values correlated with the NER capacity (P = .05) and the expression of XRCC4 (P = .01) and RAD51 (P = .06) genes., Conclusions: As revealed by the above-named assays, these lymphoblastoid cell lines derived from patients with head and neck cancer had minor alterations in DNA repair function. However, these differences in DNA repair do appear to affect the cytogenetic marker of cancer susceptibility, mutagen sensitivity.