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A new rat model was developed to reexamine the potential for laryngeal transplantation. The final anatomic derivation evolved from two earlier developmental phases. The first model had only a single arterial anastomosis; the second had an end-to-end arterial anastomosis with an end-to-end arteriovenous shunt. The final product employed an end-to-side arterial shunt and an end-to-side arteriovenous shunt for revascularization. The allografts were sited in tandem with the intact recipient larynges and were not innervated. A total of 16 animals were studied in phase 3; 2 died and the remaining 14 had a 64% arterial patency at intervals of 1 to 14 days. Our purpose is to detail the relevant technical considerations of this new model and compare it with historical controls.

Cluster of differentiation antigens (CD proteins) are classically used as immune cell markers. However, their expression within the inner ear is still largely undefined. In this study, we explored the possibility that specific CD proteins might be useful for defining inner ear cell populations. mRNA expression profiling of microdissected auditory and vestibular sensory epithelia revealed 107 CD genes as expressed in the early postnatal mouse inner ear. The expression of 68 CD genes was validated with real-time RT-PCR using RNA extracted from microdissected sensory epithelia of cochleae, utricles, saccules, and cristae of newborn mice. Specifically, CD44 was identified as preferentially expressed in the auditory sensory epithelium. Immunohistochemistry revealed that within the early postnatal organ of Corti, the expression of CD44 is restricted to outer pillar cells. In order to confirm and expand this finding, we characterized the expression of CD44 in two different strains of mice with loss- and gain-of-function mutations in Fgfr3 which encodes a receptor for FGF8 that is essential for pillar cell development. We found that the expression of CD44 is abolished from the immature pillar cells in homozygous Fgfr3 knockout mice. In contrast, both the outer pillar cells and the aberrant Deiters’ cells in the Fgfr3 P244R/ + mice express CD44. The deafness phenotype segregating in DFNB51 families maps to a linkage interval that includes CD44. To study the potential role of CD44 in hearing, we characterized the auditory system of CD44 knockout mice and sequenced the entire open reading frame of CD44 of affected members of DFNB51 families. Our results suggest that CD44 does not underlie the deafness phenotype of the DFNB51 families. Finally, our study reveals multiple potential new cell type-specific markers in the mouse inner ear and identifies a new marker for outer pillar cells.

In the vast majority of studies conducted to date, activation of cancer-specific T cell immunity through peptide-based immunization has failed to induce objective tumor regression. This failure is particularly troublesome given that these vaccines often stimulate T cell responses. In this review, we attempt to understand the relative failure of peptide cancer vaccines to achieve clinically meaningful responses. In the first part of the review, we discuss specific hurdles to successful application of synthetic peptide-based vaccines including patient variability and epitope selection. In the second part of this review, we summarize the importance of CD4+ T cell help in peptide-based vaccine strategies and offer a potential strategy to improve peptide-based vaccines through the generation of both HLA class I and class II vaccine specific-immune responses.

BACKGROUND: The purpose of this study was to determine the impact of race on outcome in patients with stage III/IV squamous cell carcinoma of the head and neck (SCCHN) who have completed concurrent chemoradiotherapy. METHODS: The authors performed a retrospective analysis of 202 patients with stage III/IV SCCHN who were treated at the University of Maryland. Patients received daily radiation to a total dose of 70.2 Gray (Gy) (1.8 Gy/day), concurrently with weekly carboplatin (area under the curve [AUC] ¼ 2) and paclitaxel (45 mg/m 2 ) chemotherapy. RESULTS: There were 108 Caucasian (CA) and 94 African American (AA) patients. The median age was 56 years, and 81% were stage IV. The median follow-up was 33 months. The median overall survival (OS) and disease-free survival (DFS) were 33 months and 19 months, respectively. When analyzed by race, the median DFS was 33 months (CA) versus 12 months (AA) (P ¼.028). The median OS was 44 months (CA) versus 24 months (AA) (P ¼.071). The 3-year DFS for stage IV AA versus stage IV CA was 29% versus 50% (P ¼.031). The 3-year DFS for N2 disease in AA versus CA was 32% versus 51% (P ¼.046). The 3-year DFS for AA versus CA with oropharyngeal tumors was 30% versus 60% (P ¼.006). CONCLUSIONS: This analysis documents the inferior outcome for AA patients. They had inferior DFS and a trend toward worse OS. When stratified by several prognostic variables, the mediocre DFS in the AA patients remains. These data suggest that further investigation into the genetic characteristics of SCCHN in AA patients is warranted. Cancer 2009;115:1744–52. V C 2009 American Cancer Society.

The ethics, justification, history, future, and rejection of laryngeal transplantation are examined in this article. The authors believe that laryngeal transplantation will be a viable alternative for a select group of patients before the end of this century.

The presence and precise structures of the glycans attached at the Fc domain of monoclonal antibodies play an important role in determining antibodies' effector functions such as antibody-dependent cell cytotoxicity (ADCC), complement activation, and anti-inflammatory activity. This paper describes a novel approach for glycoengineering of human IgG1-Fc that combines high-yield expression of human IgG1-Fc in yeast and subsequent in vitro enzymatic glycosylation, using the endoglycosidase-catalyzed transglycosylation as the key reaction. Human IgG1-Fc was first overproduced in Pichia pastoris. Then the heterogeneous yeast glycans were removed by Endo-H treatment to give the GlcNAc-containing IgG1-Fc as a homodimer. Finally, selected homogeneous glycans were attached to the GlcNAc-primer in the IgG1-Fc through an endoglycosidase-catalyzed transglycosylation, using sugar oxazolines as the donor substrates. It was found that the enzymatic transglycosylation was efficient with native GlcNAc-containing IgG1-Fc homodimer without the need to denature the protein, and the reaction could proceed to completion to give homogeneous glycoforms of IgG1-Fc when an excess of oligosaccharide oxazolines was used as the donor substrates. The binding of the synthetic IgG1-Fc glycoforms to the FcgammaIIIa receptor was also investigated. This novel glycoengineering approach should be useful for providing various homogeneous, natural or synthetic glycoforms of IgG1-Fc for structure-function relationship studies, and for future clinical applications.

Purpose: The expression of CD56, a natural killer cell–associated molecule, on αβ T lymphocytes correlates with their increased antitumor effector function. CD56 is also expressed on a subset of γδ T cells. However, antitumor effector functions of CD56+ γδ T cells are poorly characterized.Experimental Design: To investigate the potential effector role of CD56+ γδ T cells in tumor killing, we used isopentenyl pyrophosphate and interleukin-2–expanded γδ T cells from peripheral blood mononuclear cells of healthy donors.Results: Thirty to 70% of expanded γδ T cells express CD56 on their surface. Interestingly, although both CD56+ and CD56− γδ T cells express comparable levels of receptors involved in the regulation of γδ T-cell cytotoxicity (e.g., NKG2D and CD94), only CD56+ γδ T lymphocytes are capable of killing squamous cell carcinoma and other solid tumor cell lines. This effect is likely mediated by the enhanced release of cytolytic granules because CD56+ γδ T lymphocytes expressed higher levels of CD107a compared with CD56− controls following exposure to tumor cell lines. Lysis of tumor cell lines is blocked by concanamycin A and a combination of anti-γδ T-cell receptor + anti-NKG2D monoclonal antibody, suggesting that the lytic activity of CD56+ γδ T cells involves the perforin-granzyme pathway and is mainly γδ T-cell receptor/NKG2D dependent. Importantly, CD56-expressing γδ T lymphocytes are resistant to Fas ligand and chemically induced apoptosis.Conclusions: Our data indicate that CD56+ γδ T cells are potent antitumor effectors capable of killing squamous cell carcinoma and may play an important therapeutic role in patients with head and neck cancer and other malignancies.

Senescent and suppressor T cells are reported to be increased in select patients with cancer and are poor prognostic indicators. Based on the association of these T cells and poor outcomes, we hypothesized that tumors induce senescence in T cells, which negatively effects antitumor immunity. In this report, we show that human T cells from healthy donors incubated with tumor for only 6 h at a low tumor to T-cell ratio undergo a senescence-like phenotype, characterized by the loss of CD27 and CD28 expression and telomere shortening. Tumor-induced senescence of T cells is induced by soluble factors and triggers increases in expression of senescence-associated molecules such as p53, p21, and p16. Importantly, these T cells are not only phenotypically altered, but also functionally altered as they can suppress the proliferation of responder T cells. This suppression requires cell-to-cell contact and is mediated by senescent CD4+ and CD8+ subpopulations, which are distinct from classically described natural T regulatory cells. Our observations support the novel concept that tumor can induce senescent T cells with suppressor function and may effect both the diagnosis and treatment of cancer. ©2008 American Association for Cancer Research. Fil: Montes, Carolina Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Chapoval, Andrei I.. University of Maryland; Estados Unidos Fil: Nelson, Jonas. University of Maryland; Estados Unidos Fil: Orhue, Vbenosa. University of Maryland; Estados Unidos Fil: Zhang, Xiaoyu. University of Maryland; Estados Unidos Fil: Schulze, Dan H.. University of Maryland; Estados Unidos Fil: Strome, Scott E.. University of Maryland; Estados Unidos Fil: Gastman, Brian R.. University of Maryland; Estados Unidos

Several monoclonal antibodies are now in clinical use for cancer therapy, and many others are currently undergoing clinical evaluation. These agents offer unique specificity against key molecular targets on tumor cells or in the tumor microenvironment. The clinical efficacy of monoclonal antibodies is generally attributed to target-specific mechanisms resulting from neutralizing or inhibiting a growth factor or receptor that drives cell proliferation and tumor growth. Several targets, including CD20, human epidermal growth factor receptor 2, vascular endothelial growth factor, and epidermal growth factor receptor, have been validated in specific malignancies on the basis of monoclonal antibody efficacy. However, monoclonal antibodies also have the potential to activate immune-mediated effector functions, including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. These functions result from interactions involving the Fc domain of the antibody, and, consequently, may vary by antibody, isotype, and Fc modification, such as changes in glycosylation. Accordingly, all monoclonal antibodies directed against a given target should not be considered equivalent in their ability to stimulate immune-mediated effector functions.

Purpose: Human lactoferrin is a naturally occurring glycoprotein that inhibits cancer growth. Our purpose was to evaluate recombinant human lactoferrin as a chemotherapeutic agent against head and neck squamous cell carcinoma. Experimental Design: Controlled experiments both in vitro and in the murine model evaluating both the effect and mechanism of lactoferrin on cancer growth. Results: In both human and murine cell lines, lactoferrin induced dose-dependent growth inhibition. Using flow cytometric analysis, lactoferrin was shown to induce G1-G0 growth arrest. This arrest seemed to be modulated by down-regulation of cyclin D1. In the in vitro model, luminex data revealed that lactoferrin inhibited cellular release of proinflammatory and prometastatic cytokines, including interleukin-8, interleukin-6, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor-α. Lactoferrin up-regulated the cellular activation of nuclear factor-κB within 4 h of cellular exposure. In C3h/HeJ mice implanted with SCCVII tumors, orally delivered lactoferrin inhibited tumor growth by 75% compared with control mice. Immunohistochemical analysis of harvested tumors revealed up to 20-fold increases of lymphocytes within treated animals. When mice were depleted of CD3+ cells, all lactoferrin-induced tumor inhibition was abrogated. Conclusion: We conclude that human recombinant lactoferrin can inhibit the growth of head and neck squamous cell carcinoma via direct cellular inhibition as well as systemically via immunomodulation. Our data support the study of human lactoferrin as an immunomodulatory compound with therapeutic potential.

Purpose. This study was designed to evaluate the relationship between the informed consent process and the treatment received by patients with advanced laryngeal cancer. Methods. The study group was composed of 101 consecu- tive patients who were eligible for the Department of Veterans Affairs Laryngeal Cancer Study Group and Radiation Therapy Oncology Group/Head and Neck Intergroup (RTOG 91-11) lar- ynx preservation clinical trials and who underwent treatment at the Mayo Clinic during the years 1985 to 2000. Records were reviewed to evaluate the effect that referral to an oncologist, dis- cussion and documentation of treatment alternatives by the sur- geon, offering a chance at a larynx conservation surgical proce- dure, and treatment recommendation made by the surgeon have on the treatment chosen and received by the patient. Results. Discussion of treatment alternatives by the surgeon (p ¼ .0054), referral to an oncologist (p < .0001), offering a chance for a larynx conservation surgical procedure (p ¼ .047), and treatment recommended by the surgeon (p < .0001) were significantly related to the treatment received by the patient. Conclusion. The treatment received by a patient with ad- vanced laryngeal cancer is determined by the treatment recom- mended by the surgeon, referral to an oncologist, discussion of treatment alternatives by the surgeon, and offering a chance for a larynx conservation surgical procedure. V

Background. Inclusion of patients with mobile vocal cords on larynx preservation trials may lead to overstate- ment of larynx preservation (LPR) and survival (OS) rates. Methods. This is a retrospective review of patients at our institution who would have been eligible for the Department of Veterans Affairs Laryngeal Cancer Study Group (VA) and Radiation Therapy Oncology Group/Head and Neck Intergroup (RTOG 91-11) clinical trials. No laryngeal conservation proce- dure could be performed in patients with mobile vocal cords. Results. One hundred one patients at our institution would have been eligible for the VA trial. The 2-year OS was 76% for total laryngectomy and 90% for radiotherapy patients (p ¼ .28) compared with 68% reported for the VA trial. Seventy-three patients at our institution would have been eligible for the RTOG 91-11 trial. The 5-year OS was 52% for total laryngectomy and 63% for radiotherapy patients (p ¼ .18) compared with 55% reported for the RTOG 91-11 trial. Radiotherapy patients had an LPR of 80% (VA trial) and 86% (RTOG 91-11 trial). Conclusion. It is unlikely that inclusion of patients with mobile vocal cords had a significant impact on OS or LPR for the VA and RTOG 91-11 larynx preservation trials. V V C 2006 Wiley Periodicals, Inc. Head Neck 28: 689-696, 2006

Objectives/Hypothesis: The objectives of this study were to delineate methods for the development of primary squamous cell carcinoma (SCCHN) xenografts and to define human leukocyte antigen (HLA), melanoma-associated antigen (MAGE)-A3, and human papilloma virus (HPV) 16 antigenic expression in resultant cellular products. Study Design: Prospective experimental model. Methods: Freshly isolated SCCHN xenografts were established in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice using a variety of methods. Resultant tumors were analyzed for expression patterns of HLA-A, MAGE-A3, and HPV 16. Appropriate controls were included to ensure the presence of human RNA. Results: Three xenografts were successfully established and passaged in vivo. Characterization of the resultant products revealed that one was positive for HLA-A2 at both the DNA and protein levels. One of the tumor lines expressed MAGE-A3, whereas none expressed HPV 16. Conclusions: Freshly isolated SCCHN can be used to generate primary xenografts. Characterization of select patterns of protein expression in established xenografts is a precursor to the development of a mouse model for SCCHN using tumor bearing animals reconstituted with autologous patient leukocytes.

BACKGROUND The most common long-term complication of radiation therapy (RT) for head and neck cancer is dry mouth or xerostomia. Salivary glands are radiosensitive; their destruction begins shortly after the initiation of therapy with the extent of injury directly related to the amount of radiation to which they are exposed. The subsequent xerostomia is challenging to manage, leads to multiple and often long-term oral complications, and can significantly impair patients’ quality of life. This article utilizes comprehensive review of literature, identified through publicly available search engines, to outline the current best practice recommendations for patients with RT-induced xerostomia.

Expression of B7-H1, a costimulating glycoprotein in the B7 family, is normally restricted to macrophage-lineage cells, providing a potential costimulatory signal source for regulation of T cell activation. In contrast, aberrant expression of B7-H1 by tumor cells has been implicated in impairment of T cell function and survival, resulting in defective host antitumoral immunity. The relationship between tumor-associated B7-H1 and clinical cancer progression is unknown. Herein, we report B7-H1 expression by both renal cell carcinoma (RCC) tumors of the kidney and RCC tumor-infiltrating lymphocytes. In addition, our analysis of 196 clinical specimens reveals that patients harboring high intratumoral expression levels of B7-H1, contributed by tumor cells alone, lymphocytes alone, or tumor and/or lymphocytes combined, exhibit aggressive tumors and are at markedly increased risk of death from RCC. In fact, patients with high tumor and/or lymphocyte B7-H1 levels are 4.5 times more likely to die from their cancer than patients exhibiting low levels of B7-H1 expression (risk ratio 4.53; 95% confidence interval 1.94–10.56; P < 0.001.) Thus, our study suggests a previously undescribed mechanism whereby RCC may impair host immunity to foster tumor progression. B7-H1 may prove useful as a prognostic variable for RCC patients both pre- and posttreatment. In addition, B7-H1 may represent a promising target to facilitate more favorable responses in patients who require immunotherapy for treatment of advanced RCC.

Objectives: Total maxillectomy creates a significant defect in the supporting framework of the orbit. Successful reconstruction of the deficit requires repair of the orbital floor to prevent early and late complications. This paper describes the recreation of the orbital floor using a coronoid-temporalis sling. Study Design: Retrospective review of two patients who underwent total maxillectomy and subsequent coronoid-temporalis sling reconstruction of the surgical defect. Methods: The charts of two patients who underwent coronoid-temporalis reconstruction of the orbital floor were retrospectively reviewed. The outcomes were evaluated. The authors present a method for reconstruction of the orbital floor and discuss the advantages and disadvantages of this method. Results: Two patients underwent successful reconstruction of the orbital floor with a coronoid-temporalis sling procedure. Both patients display acceptable functional and cosmetic results. Conclusion: Coronoid-temporalis sling is a readily available and easily modified tissue for reconstruction of the orbital floor. It offers similar results to those previously described techniques in this area without need for further extensive surgery.

Manipulation of defined costimulatory pathways may provide a means to direct the immune response against head and neck cancer. Three broad hurdles serve as relative obstacles to effective clinical translation of reagents designed to alter costimulatory function. First is an expanding, but still limited, understanding of the biological function of each individual costimulatory pathway. Second is the paucity of reagents suitable for clinical application. Third is the limited number of valid model systems to evaluate the utility of these novel reagents prior to clinical intervention. This review focuses on the role of a recently discovered costimulatory molecule, B7-H1, as a model for the development of novel strategies for immunotherapy of head and neck cancer.

Squamous cell carcinoma of the head and neck (SCCHN) is an important source of cancer-related morbidity and mortality worldwide. Current treatment options are founded on various combinations of surgery, radiation therapy, and chemotherapy, with implementation largely based on patient/disease-specific factors and provider/institutional bias. Despite improvements in surgical techniques and the development of novel chemoradiotherapy strategies for organ preservation, survival has remained relatively unchanged in the past 30 years. Additionally, the impact of these novel treatment approaches on the quality of life of patients remains largely unknown. Improvements in survival and the quality of life for patients with SCCHN will likely require the development of new therapeutic alternatives based on the improved understanding of the molecular pathogenesis of SCCHN and associated changes in the immune response.

In this report, we demonstrate that B7-H1, a B7 family molecule implicated in tumor immune evasion, is constitutively expressed on 66% of freshly isolated squamous cell carcinomas of the head and neck (SCCHN). To define the potential impact of tumor-associated B7-H1 on immunotherapy, the B7-H1-negative mouse SCC line, SCCVII, was transfected to express B7-H1. Although all of the animals succumbed to B7-H1/SCCVII tumors even after adoptive T-cell immunotherapy, the infusion of B7-H1 blocking monoclonal antibody with activated T cells cured 60% of animals. These data support B7-H1 blockade as a new approach to enhance the efficacy of T-cell immunotherapy.

Autoimmune Inner Ear Disease (AIED) is a clinical syndrome of uncertain pathogenesis. It is associated with bilateral rapidly progressive hearing loss. The hearing loss may be associated with vestibular symptoms. Autoimmunity has been proposed as the pathogenesis of this sort of hearing loss, although the mechanism of the disease is poorly understood. It is well accepted that the endolymphatic sac is an immunocompetent organ and circulating autoantibodies against inner ear antigens have been reported, as have viral antigens in the endolymph, although the sensitivity, specificity, and roles of those antibodies in a disease process are poorly defined. We will describe the clinical aspects of the disease, the histopathology, the immunologic indicators, the types of presentation, both from the audiologic and vestibular point of view, clinical trials for treatment and the follow-up. One of our conclusions is that many of these patients respond favorably to the treatment Methotrexate.

Objectives To determine the prevalence of nodal metastases in the submuscular recess (SMR) in patients undergoing selective neck dissection (SND) and to identify potential risk factors for the presence of metastatic disease in the SMR. Design Prospective cohort study. Setting Academic tertiary care referral center. Patients Consecutive patients undergoing SND for squamous cell carcinoma of the head and neck between January 5, 1998, and November 23, 2001, were prospectively analyzed. Patients with a history of neck dissection or whose pathology reports did not clearly distinguish the SMR from other nodal levels were excluded from the study. Interventions Patients underwent SND based on the primary tumor site and well established regional lymphatic drainage patterns. Main Outcome Measure Presence of histopathologically proven nodal disease in the SMR. Results Seventy-four patients underwent 90 SNDs, 16 of which were bilateral. The prevalence of metastases in the SMR was 1.6% (1/63) in clinically N0 necks and 11.1% (3/27) in clinically node-positive necks, with an overall incidence of 4.4% (4/90). There was a statistically significant association between SMR metastases and advanced pathologic N stage (P= .003), particularly with positive nodal disease in level IIa (P= .001). Extracapsular tumor spread was also shown to have a statistically significant association with metastases in the SMR (P= .01). No significant associations were observed between SMR metastases and primary tumor site (P= .06), clinical N stage (P= .09), a history of primary tumor recurrence (P= .52), or previous radiation therapy (P= .68). Conclusion The results of the present study suggest that nodal metastases in the SMR are rare in head and neck cancer patients undergoing SND.

Background. Certain tumor antigens have been identified that stimulate an immune response, thus making them targets for immunotherapy. NY-ESO-1, MAGE-1, and MAGE-3 are such antigens. This study was undertaken to determine their presence or absence in head and neck squamous cell cancers and to correlate this with patient characteristics. Methods. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry (IH) were used to identify NY-ESO-1, MAGE-1, and MAGE-3 in surgical specimens. Patient data (previous treatment, gender, age, primary site, metastasis, tumor grade, tumor stage, smoking history, and alcohol history) were collected by chart review and examined for correlation with presence or absence of antigen. Results. Three tumors were found to be positive for NY-ESO-1 by RT-PCR. All of these tumors were also positive for MAGE-1 and MAGE-3. IH was only positive for NY-ESO-1 in one patient. Eighteen of the 45 tumors (40%) were positive for MAGE-1 by RT-PCR. By IH, only six tumors were positive for MAGE-1. Five (83.3%) of those that were positive by IH were positive by RT-PCR. Twenty of the 45 tumors (44.4%) were positive for MAGE-3 by RT-PCR. By IH, 12 tumors were positive for MAGE-3. Nine (75%) of those positive by IH were also positive by RT-PCR. Overall, of the 45 tumors, 27 (60%) were positive by RT-PCR for at least one of the antigens. None of the patient characteristics correlated with the presence or absence of antigen. Conclusions. There is high expression of MAGE-1 and MAGE-3 antigens in head and neck squamous cell carcinomas, whereas NY-ESO-1 is not significantly expressed. IH correlates but is not as sensitive as RT-PCR for detection of these antigens. There is no correlation between antigen expression and patient data. On the basis of the high levels of MAGE-1 and MAGE-3 expression, use of these antigens may serve as a potential approach to immunotherapy for squamous cell carcinoma from head and neck sources. © 2003 Wiley Periodicals, Inc. Head Neck 25: 457–463, 2003

Background. Eighty-five percent of all Merkel cell carcinomas appear on sun-exposed areas, with 50% to 55% occurring on the head and neck. Methods. A chart review was performed on 22 patients treated for Merkel cell carcinoma of the head and neck between 1981 and 1998. Results. Fifteen patients were men (68%). The average age at operation was 69.9 years (range, 24-84 years). The average duration of follow-up was 3.6 years (range, 3 days-8.6 years). Overall survival at 1, 2, and 3 years postoperatively was 78%, 68%, and 68%, respectively. The only independent predictor of survival was the type of surgical therapy. All patients who underwent wide local excision (WLE) of the primary tumor with dissection of the lymphatic drainage basin were alive at 2 years as opposed to 68% who had WLE alone and 33% who had Mohs surgery. Conclusions. WLE and dissection of the lymphatic drainage basin provided the best overall survival.

Although neuroblastoma is the most common of extracranial solid tumors of childhood and infancy, we report the first case of an isolated neuroblastoma of a paranasal sinus. A 15-year-old girl with a right maxillary sinus mass was asymptomatic except for persistent epiphora. Computed tomography and magnetic resonance imaging scans showed that the mass extended into the nasal cavity, encroached on the lamina papyracea, and obstructed the nasofrontal duct. An extensive workup revealed no evidence of systemic disease. The patient underwent right craniofacial resection. Immunohistochemistry and electronmicroscopic findings were consistent with conventional neuroblastoma. Fluorescence in situ hybridization analysis was performed with probes selected to demonstrate genetic alterations associated with neuroblastoma. Studies revealed deletion of chromosome arm 1p, gain of chromosome 17, and normal N-myc gene copy number. In summary, the tumor exhibited morphologic features and genetic alterations more consistent with those of neuroblastoma than with those of esthesioneuroblastoma.

The role of human papilloma virus (HPV) in the pathogenesis and biological behavior of tonsillar squamous cell carcinoma (TSCC) are areas of intense investigation.This study used PCR analysis to identify HPV in paraffin-embedded tonsillar and nodal tissue from 52 patients with TSCC and 48 age (+/-5 year)/gender-matched controls with benign tonsillar hyperplasia. Results were correlated with HLA-DRB1 haplotype and clinical outcome.HPV was identified in 46% of patients with TSCC and 6% of controls. DNA sequencing showed the presence of HPV type 16 in 21 patients (40%) with TSCC. There was no statistically significant association between HLA-DRB1 expression and TSCC or HPV infection. Fifteen of 16 patients with HPV-positive TSCC with regional metastases had evidence of HPV in pathologically involved lymph nodes. In eight HPV 16-positive TSCC patients with lymph node metastasis, PCR testing identified HPV 16 in 17 of 23 histologically negative lymph nodes. Patients with HPV-positive TSCC without metastatic disease had no evidence of HPV in their lymphatic tissue. Clinically, HPV-associated carcinoma was present in younger patients in comparison with HPV-negative TSCC patients (mean age, 56.6 versus 66 years; P = 0.001). The odds for patients with HPV infection to develop TSCC were 18.2 times greater than for patients without HPV infection (95% confidence interval 4.6, 73.1). There was no statistically significant association between presence of HPV and cause-specific survival (hazard ratio = 2.5 for HPV negative versus positive; P = 0.26), after adjusting for age in a Cox proportional hazards regression analysis.HPV is an independent risk factor for TSCC. Identification of HPV in the histologically positive and negative lymph nodes of patients with HPV-positive TSCC/metastatic disease supports the role of HPV in the oncogenesis of TSCC.

Dendritic cells (DCs) primed with tumor antigens can effectively mediate the regression of a variety of established solid malignancies in both murine and human models. Despite such clinical efficacy, the optimal means of DC priming is unknown. The goal of this study was to compare three methods of tumor preparation: irradiation, boiling, or freeze thaw lysis for DC priming. Mouse bone marrow-derived DCs were loaded with defined ratios of E.G7 tumor cells expressing a model tumor antigen, OVA. Sensitized DCs were used for stimulation of OVA-specific CTLs derived from OT-1 T-cell receptor transgenic mice. IFN-gamma release, determined by ELISA at 24 and 48 h, was used to assess the expression of antigens by DCs. DCs loaded with irradiated tumors were effective stimulators for OT-1 CTLs, whereas DCs stimulated with freeze-thawed or boiled tumors did not stimulate IFN-gamma production. Freeze-thaw lysis appeared to inhibit CTL activity in vitro and in two of three cases, this effect was not overcome by the addition of OVA. The ability to load irradiated tumor cells was reproduced in two analogous human melanoma models using melanoma cell lines expressing gp100 and CTL clones specific for a gp100 melanoma antigen. Consistent with the in vitro data, only DC/irradiated tumor vaccines were effective in preventing or delaying outgrowth of E.G7 and a poorly immunogenic murine squamous cell carcinoma (SCCVII), on local tumor challenge. These data demonstrate that the method of tumor cell preparation clearly influences the ability of DCs to present antigen to T cells. Correlation of in vitro data with the generation of protective immunity in vivo suggests the utility of irradiated tumor-primed DCs as a means to generate protective immunity in patients with solid malignancies.

Treatment of advanced, poorly immunogenic tumors in animal models, considered the closest simulation available thus far for conditions observed in cancer patients, remains a major challenge for cancer immunotherapy. We reported previously that established tumors in mice receiving an agonistic mAb to the T cell costimulatory molecule 4-1BB (CD137) regress due to enhanced tumor antigen-specific cytotoxic T lymphocyte responses. In this study, we demonstrate that several poorly immunogenic tumors, including C3 tumor, TC-1 lung carcinoma, and B16-F10 melanoma, once established as solid tumors or metastases, are refractory to treatment by anti-4-1BB mAb. We provide evidence that immunological ignorance, rather than anergy or deletion, of tumor antigen--specific CTLs during the progressive growth of tumors prevents costimulation by anti-4-1BB mAb. Breaking CTL ignorance by immunization with a tumor antigen-derived peptide, although insufficient to stimulate a curative CTL response, is necessary for anti--4-1BB mAb to induce a CTL response leading to the regression of established tumors. Our results suggest a new approach for immunotherapy of human cancers.

Receptors for interleukin 4 (IL-4R) are overexpressed on the surface of various human solid tumors including renal cell carcinoma, glioblastoma, Kaposi's sarcoma, and head and neck squamous cell carcinoma (SCCHN). On the basis of this preferential receptor overexpression, a novel IL-4R-targeted cytotoxin, IL-4 (38-37)-PE38KDEL, was developed in which circularly permuted IL-4 [IL-4 (38-37)] was fused to mutated form of Pseudomonas exotoxin (PE38KDEL). Despite the recognized expression of the IL-4R on SSCHN, the utility of a receptor-specific fusion protein for the treatment of this disease remains unknown. The purpose of this study was to establish the utility of IL-4 (38-37)-PE38KDEL for the treatment of established SSCHN in animal models of human disease. Expression of IL-4R in SCCHN was confirmed by immunohistochemistry with eight of eight tissue sections expressing IL-4R. Protein synthesis inhibition assays demonstrated growth inhibition of two cell lines in IL-4 (38-37)-PE38KDEL in a dose-dependent fashion. In two SCCHN s.c. xenografted nude mouse models, i.p. and intratumoral injection of IL-4 (38-37)-PE38KDEL mediated tumor regression with no visual toxicity observed in any of the animals. Subcultured tumor cells after intratumoral treatment with IL-4 toxin did not develop resistance to the drug. These data demonstrate that IL-4 (38-37)-PE38KDEL is effective in mediating significant antitumor effects in SCCHN and may represent an attractive therapeutic option for patients with advanced cancers of the upper aerodigestive tract.

Importance Noncongenital immature teratomas in the head and neck are extremely rare, and to our knowledge have not previously been reported in the maxillary sinus of a pediatric patient. Observations Teratomas are neoplasms that contain tissue from more than 1 embryonic cell layer that is foreign to the location where they arise and are most commonly found in the sacrococcygeal and gonadal regions in children. We present an unusual case of an immature teratoma in the maxillary sinus of a 10-year-old boy and discuss the diagnostic workup, management, and clinical course. Conclusions and Relevance Immature teratomas represent a subset of benign teratomas with immature elements that may carry a worse prognosis.

e17014 Background: New therapeutic modalities are needed for RM-SCCHN. We conducted a phase I dose escalation study to evaluate the safety and immunologic response to peptide immunomodulatory vacci...

6083 Background: Racial outcome disparities have been observed in head and neck cancer with diminished survival for black patients compared to whites. The etiology of this disparity appears to be m...

Background To develop novel therapeutic approaches for patients with head and neck malignancies, poorly immunogenic murine models of squamous cell carcinoma (SCC) need to be defined. Methods The phenotype, growth characteristics, and responsiveness to tumor-specific T-cell transfer of a spontaneously arising murine SCC (SCC VII) were characterized. Results SCC VII expresses major histocompatibility complex (MHC) class I molecules yet is resistant to tumor-specific T-cell killing and relatively insensitive to killing mediated by lymphokine-activated killer (LAK) cells. Intradermal tumors are reproducibly established after vaccination of 5 × 104 cells, and systemic micrometastases are apparent after intravenous administration of 2.5 × 104 cells. Immunotherapy of 3-day lung metastases using tumor-specific T cells and systemic interleukin-2 (IL-2) was ineffective in reducing the number of metastases in vivo. Conclusions SCC VII is a poorly immunogenic murine squamous cell cancer, which represents an ideal model for preclinical testing of immunotherapeutic approaches for patients with SCC of the upper aerodigestive tract. © 2001 John Wiley & Sons, Inc. Head Neck 23: 899–906, 2001.

Epstein-Barr virus (EBV) is a known oncogenic virus associated with a wide variety of cancers, including nasopharyngeal carcinoma. Waldeyer's ring, a collection of lymphoid tissues, includes the nasopharynx, pharyngeal, and lingual tonsils. To determine if EBV plays a causative role in carcinomas arising from other tissues in Waldeyer's ring, we examined pharyngeal tonsillar carcinomas for evidence of EBV infection. As previously reported, DNA was extracted from 53 consecutive tonsil cancers, as well as from age- and gender-matched non-cancerous tonsillectomy specimens. Three different sets of primers for discrete exons of EBV were then used to determine if active or latent EBV infection was expressed in the extracted DNA using the polymerase chain reaction (PCR). All positive bands were then sequenced to confirm the presence of amplified EBV fragments. None of the samples showed evidence for active EBV infection. In primers demonstrating latent infection, 1 of 53 (1.9%) of tumors were positive, versus 6 of 53 (11.3%) of the controls. These results indicate that EBV expression is not increased in DNA from tonsil cancers and that EBV infection does not have a causal relationship with tonsil cancer.

On February 10, 1969, Kluyskens performed a partial laryngeal transp1antation.l Although initial graft viability and function proved such an operation to be feasible, secondary failure due to cancer recurrence highlighted the inherent conflict between “acceptable risk,” both to the individual and society, and “potential benefit.” When considering the potential for laryngeal transplantation today, the dilemma that otolaryngologists and society as a whole must confront still revolves around these fundamental issues. Recent advances in immunosuppression, graft preservation techniques, and an understanding of the cellular mechanisms of allograft rejection have diminished the risk of this therapeutic modality and warrant reconsideration of the ethics of laryngeal transplantation in the context of current scientific knowledge. A prerequisite for any type of ethical analysis of laryngeal transplantation is a statement of purpose. The goals of this procedure are the restoration of near-normal speech, deglutition, and cosmesis with a minimal risk of aspiration. In order to attain these benefits, the patient accepts significant risks, including a potentially increased probability of cancer recurrence, graft rejection, and the inherent morbidity of chronic immunosuppression. If we are to conclude that this procedure is ethical, it is first necessary to ascertain that such goals cannot be achieved by currently available surgical or medical techniques with less patient risk. There are several operative techniques for voice restoration in the post-laryngectomy pa

Purpose: The effect of high-dose radiation therapy on facial nerve grafts is controversial. Some authors believe radiotherapy is so detrimental to the outcome of facial nerve graft function that dynamic or static slings should be performed instead of facial nerve grafts in all patients who are to receive postoperative radiation therapy. Unfortunately, the facial function achieved with dynamic and static slings is almost always inferior to that after facial nerve grafts. In this retrospective study, we compared facial nerve function in irradiated and unirradiated nerve grafts. Methods and Materials: The medical records of 818 patients with neoplasms involving the parotid gland who received treatment between 1974 and 1997 were reviewed, of whom 66 underwent facial nerve grafting. Fourteen patients who died or had a recurrence less than a year after their facial nerve graft were excluded. The median follow-up for the remaining 52 patients was 10.6 years. Cable nerve grafts were performed in 50 patients and direct anastomoses of the facial nerve in two. Facial nerve function was scored by means of the House–Brackmann (H-B) facial grading system. Twenty-eight of the 52 patients received postoperative radiotherapy. The median time from nerve grafting to start of radiotherapy was 5.1 weeks. The median and mean doses of radiation were 6000 and 6033 cGy, respectively, for the irradiated grafts. One patient received preoperative radiotherapy to a total dose of 5000 cGy in 25 fractions and underwent surgery 1 month after the completion of radiotherapy. This patient was placed, by convention, in the irradiated facial nerve graft cohort. Results: Potential prognostic factors for facial nerve function such as age, gender, extent of surgery at the time of nerve grafting, preoperative facial nerve palsy, duration of preoperative palsy if present, or number of previous operations in the parotid bed were relatively well balanced between irradiated and unirradiated patients. However, the irradiated graft group had a greater proportion of patients with pathologic evidence of nerve invasion ( p = 0.007) and unfavorable type of nerve graft ( p = 0.04). Although the irradiated graft cohort had more potentially negative prognostic factors, there was no difference in functional outcome (H-B Grade III or IV) between irradiated and unirradiated graft patients. H-B Grades III, IV, V, and VI were the best postoperative facial nerve functions achieved in 35%, 39%, 13%, and 13% of patients, respectively. The patient with preoperative radiotherapy never recovered any facial nerve function (H-B Grade VI). Median time to best facial nerve function after surgery was longer in the irradiated patients (13.1 vs. 10.8 months), but this was not statistically significant ( p = 0.10). Presence of preoperative facial nerve palsy ( p = 0.005), duration of preoperative palsy ( p = 0.003), and age greater than 60 years at the time of grafting ( p = 0.04) were all negative prognostic factors for achieving a functional facial nerve on univariate analysis. Analysis of age as a continuous variable ( p = 0.12) and pathologic evidence of nerve invasion ( p = 0.1) revealed a trend toward negative prognostic factors. Gender, number of previous operations in the parotid bed, extent of surgery at the time of nerve grafting, and type of grafting procedure were not significant prognostic factors. Whether radiotherapy was delivered less than 6 weeks after nerve grafting or more than 6 weeks had no impact on achievement of a functional facial nerve. Conclusion: Negative prognostic factors for achieving a functional facial nerve in our series include the presence of preoperative facial nerve palsy, duration of preoperative palsy, and age greater than 60 years. Radiotherapy was not a negative prognostic factor. Comparing irradiated and unirradiated grafts revealed no difference in best facial nerve function achieved, despite the presence of a greater proportion of negative prognostic factors in the irradiated group. Therefore, planned postoperative radiation therapy is not a contraindication to facial nerve grafting. Consideration for regeneration of the facial nerve should not influence the timing of postoperative radiotherapy, because early initiation of radiotherapy after facial nerve grafting did not have a deleterious effect on facial nerve function. However, the time required to attain best facial nerve function postoperatively may be slightly longer in irradiated patients.

Costimulation plays a critical role in T-cell activation and amplification of anti-tumor immunity. Although CD28 engagement triggers an early activation signal, activation-induced 4-1BB molecule on T cells transmits a crucial signal for further expansion and maturation of effector cells. In this report, the authors show that costimulation through CD28 and 4-1BB pathways synergistically enhances the therapeutic efficacy of T cells from tumor-draining lymph nodes. Intravenous adoptive transfer of costimulated T cells into mice bearing disseminated micrometastasis of a poorly immunogenic, major histocompatibility complex class I-negative A9P melanoma results in a 60% cure rate. Autopsy of mice that died after unsuccessful treatment revealed tumor growth in the liver, spleen, and skin with minimal or no evidence of pulmonary disease. In contrast, mice that received no treatment or noncostimulated T cells had massive pulmonary tumors, suggesting that adoptively transferred T cells are less effective against growth of extrapulmonary tumors. These results show that costimulation of tumor-draining lymph node T cells through CD28 and 4-1BB increases their potential for cancer immunotherapy and suggests that improper trafficking of tumor-reactive T cells to extrapulmonary sites must be improved to enhance clinical efficacy.

The interaction of Fc fragments of antibodies with the Fcgamma receptors is an essential checkpoint in antibody-dependent cellular cytotoxicity (ADCC). Specific polymorphisms at position 158 enhance FcgammaRIIIa affinity for IgG1 and are associated with improved clinical outcome in lymphoma patients treated with IgG1 anti-CD20 antibody. The role of ADCC in the therapeutic effects of the alpha-epidermal growth factor receptor (EGFR) mAb, cetuximab, in patients with squamous cell carcinoma of the head and neck (SCCHN) is poorly defined. We employed three SCCHN cell lines to test two hypotheses: (1) SCCHN is susceptible to cetuximab-mediated ADCC, (2) efficacy of ADCC is associated with polymorphisms at position 158 of FcgammaRIIIa.FcgammaRIIIa-158 polymorphisms were determined for healthy donors, and their purified NK cells were used as effector cells against three SCCHN cell lines in ADCC assays. Cytotoxicity levels were compared for each polymorphism class. Proliferation and cell cycle assays were done to examine the direct effects of cetuximab.Our results indicate that SCCHN is susceptible to cetuximab-mediated ADCC in vitro. NK cytotoxic efficiency correlates with donor 158-polymorphisms in FcgammaRIIIa. Overall cytotoxicity was greatest for individuals having a single V allele when compared to homozygous F/F individuals; the cumulative percent cytotoxicity for each polymorphism among the cell lines was 58.2% V/V, 50.6% V/F, and 26.1% F/F (P0.001). Additionally, the presence of a V allele correlated with superior natural cytotoxicity against NK sensitive targets.These data have both prognostic and therapeutic relevance and support the design of a prospective trial to determine the influence of FcgammaRIIIa polymorphisms on the clinical outcome of patients with SCCHN treated with alpha-EGFR mAbs.

The purpose of this study was to establish treatment criteria for patients with early-stage squamous cell carcinoma of the buccal mucosa. Thirty-one patients were analyzed in a retrospective fashion. Distribution of patients according to tumor stage was relatively even. Within 5 years recurrent disease developed in nearly 80% of evaluable patients. There was a 100% overall incidence of local disease recurrence for patients with stage I and II tumors treated with wide local excision alone and followed up for more than 2 years. On the basis of these data, we conclude that wide local excision for early-stage buccal carcinoma is associated with a high local failure rate. Possible causes for failure and alternative treatment approaches are discussed.

The generation of a therapeutic immune response to malignancy is critically dependent on the inherent immunogenicity of the tumor. Our study demonstrates that secretion of both interleukin-2 (IL-2) and IL-4 by a seemingly nonimmunogenic tumor abrogates tumorigenicity, and mice that have rejected the genetically modified tumor are immune to challenges with the parental tumor. The induction of immunity by the IL-2/IL-4-secreting tumor was significantly better than that achieved with the admixture of tumor cells and the classic adjuvant, Corynebacterium parvum. To elicit a primary immune response, the majority of cells needed to secrete both cytokines. Ad-mixture of IL-2-secreting cells with IL-4-secreting cells did not result in tumor cell rejection. The IL-2/IL-4-secreting tumor cells were efficiently rejected in animals immunosuppressed by total body irradiation. Depletion of CD4+ or CD8+ T cells did not abrogate rejection of the tumor cells, but the animals depleted of CD4 cells failed to generate protective immunity. Our study demonstrates that secretion of the combination of IL-2 and IL-4 significantly enhances tumor immunogenicity. The requirement of cells secreting both cytokines suggests an intricate mechanism different from the mere presence of both cytokines at the tumor-inoculation site.

Laryngeal transplantation was actively investigated in the late 1960s and early 1970s using a dog model. An expanded knowledge base in immunobiology, pharmacology, and deglutition makes reassessment timely after an approximately 20-year hiatus. The basic parameters to be evaluated include varied methods of preservation, optimal immunosuppressive drug regimens, and the role of radiation therapy. Our current research protocol addressing the latter considerations is outlined, including a brief introduction of a new animal model developed specifically for this purpose. We will ultimately attempt to answer the question: Is laryngeal transplantation feasible?

Heterozygous Twirler (Tw) mice develop obesity and circling behavior associated with malformations of the inner ear, whereas homozygous Tw mice have cleft palate and die shortly after birth. Zeb1 is a zinc finger protein that contributes to mesenchymal cell fate by repression of genes whose expression defines epithelial cell identity. This developmental pathway is disrupted in inner ears of Tw/Tw mice. The purpose of our study was to comprehensively characterize the Twirler phenotype and to identify the causative mutation. The Tw/+ inner ear phenotype includes irregularities of the semicircular canals, abnormal utricular otoconia, a shortened cochlear duct, and hearing loss, whereas Tw/Tw ears are severely malformed with barely recognizable anatomy. Tw/+ mice have obesity associated with insulin-resistance and have lymphoid organ hypoplasia. We identified a noncoding nucleotide substitution, c.58+181G>A, in the first intron of the Tw allele of Zeb1 (Zeb1Tw). A knockin mouse model of c.58+181G>A recapitulated the Tw phenotype, whereas a wild-type knockin control did not, confirming the mutation as pathogenic. c.58+181G>A does not affect splicing but disrupts a predicted site for Myb protein binding, which we confirmed in vitro. In comparison, homozygosity for a targeted deletion of exon 1 of mouse Zeb1, Zeb1ΔEx1, is associated with a subtle abnormality of the lateral semicircular canal that is different than those in Tw mice. Expression analyses of E13.5 Twirler and Zeb1ΔEx1 ears confirm that Zeb1ΔEx1 is a null allele, whereas Zeb1Tw RNA is expressed at increased levels in comparison to wild-type Zeb1. We conclude that a noncoding point mutation of Zeb1 acts via a gain-of-function to disrupt regulation of Zeb1Tw expression, epithelial-mesenchymal cell fate or interactions, and structural development of the inner ear in Twirler mice. This is a novel mechanism underlying disorders of hearing or balance., Author Summary Twirler (Tw) mice have a combination of abnormalities that includes cleft palate, malformations of the inner ear, hearing loss, vestibular dysfunction, obesity, and lymphoid hypoplasia. In this study, we show that the underlying mutation affects the Zeb1 gene. Zeb1 was already known to encode a protein normally expressed in mesenchymal cells, where it represses expression of genes that are uniquely expressed in epithelial cells. The Tw mutation is a rare example of a single-nucleotide substitution in a region of a gene that does not encode protein, promoter, or splice sites, so we engineered a mouse model with the mutation that confirmed its causative role. The Tw mutation disrupts a consensus DNA binding site sequence for the Myb family of regulatory proteins. We conclude that this mutation leads to abnormal expression of Zeb1, structural malformations of the inner ear, and a loss of hearing and balance function. A similar mechanism may underlie other features of Twirler, such as obesity and cleft palate.

Background The extracellular signal-regulated kinase-1 and 2 (ERK1/2) proteins play an important role in cancer cell proliferation and survival. ERK1/2 proteins also are important for normal cell functions. Thus, anti-cancer therapies that block all ERK1/2 signaling may result in undesirable toxicity to normal cells. As an alternative, we have used computational and biological approaches to identify low-molecular weight compounds that have the potential to interact with unique ERK1/2 docking sites and selectively inhibit interactions with substrates involved in promoting cell proliferation. Methods Colony formation and water soluble tetrazolium salt (WST) assays were used to determine the effects of test compounds on cell proliferation. Changes in phosphorylation and protein expression in response to test compound treatment were examined by immunoblotting and in vitro kinase assays. Apoptosis was determined with immunoblotting and caspase activity assays. Results In silico modeling was used to identify compounds that were structurally similar to a previously identified parent compound, called 76. From this screen, several compounds, termed 76.2, 76.3, and 76.4 sharing a common thiazolidinedione core with an aminoethyl side group, inhibited proliferation and induced apoptosis of HeLa cells. However, the active compounds were less effective in inhibiting proliferation or inducing apoptosis in non-transformed epithelial cells. Induction of HeLa cell apoptosis appeared to be through intrinsic mechanisms involving caspase-9 activation and decreased phosphorylation of the pro-apoptotic Bad protein. Cell-based and in vitro kinase assays indicated that compounds 76.3 and 76.4 directly inhibited ERK-mediated phosphorylation of caspase-9 and the p90Rsk-1 kinase, which phosphorylates and inhibits Bad, more effectively than the parent compound 76. Further examination of the test compound's mechanism of action showed little effects on related MAP kinases or other cell survival proteins. Conclusion These findings support the identification of a class of ERK-targeted molecules that can induce apoptosis in transformed cells by inhibiting ERK-mediated phosphorylation and inactivation of pro-apoptotic proteins.

This study used both clinical and histopathologic criteria to define the sequence and time parameters of rejection in histoincompatible vascularized rat laryngeal allografts. Clinical onset of rejection was characterized at 1 week by graft edema. Pathologic examination at this time revealed arterial wall thickening and a diffuse lymphocytic and macrophage mucosal infiltrate. At 14 days, the graft was encased in an inflammatory exudate with no visible cartilaginous structure. This correlated microscopically with a lymphocytic infiltrate of the lamina propria, loss of minor salivary glands, and squamous metaplasia of the surface respiratory epithelium. Arterial thrombosis was always correlated with tissue necrosis, but venous occlusion, occurring more than 2 days after transplantation, was not an independent determinant of graft viability. Definition of the time sequence and histopathology of rejection will allow future determination of the efficacy of various immunosuppressive regimens.