Clinical experience with HLA-B7 plasmid DNA/lipid complex in advanced squamous cell carcinoma of the head and neck.

OBJECTIVE: To investigate the safety and efficacy of alloantigen plasmid DNA therapy in patients with advanced head and neck squamous cell carcinoma using Allovectin-7 (Vical Inc, San Diego, Calif), a DNA/lipid complex designed to express the class I major histocompatibility complex antigen HLA-B7. DESIGN: Multi-institutional prospective trial. SETTING: Academic medical setting. PATIENTS: A total of 69 patients were enrolled in 3 sequential clinical trials: a single-center phase 1 trial and 2 multicenter phase 2 trials. Eligibility criteria included unresectable squamous cell carcinoma that failed conventional therapy, Karnofsky performance status score of 70 or greater, and no concurrent anticancer or immunosuppressive therapies. INTERVENTION: Patients received 2 biweekly intratumoral injections of 10 microg (phase 1 and first phase 2 trials) or 100 microg (second phase 2 trial) of Allovectin-7 followed by 4 weeks of observation. Patients with stable or responding disease after the observation period were given a second treatment cycle identical to the first. MAIN OUTCOME MEASURES: Patients were assessed for toxic effects, and tumor size was measured after cycles 1 (at 6 weeks) and 2 (at 16 weeks). RESULTS: Allovectin-7 treatment was well tolerated, with no grade 3 or 4 drug-related toxic effects. Of 69 patients treated, 23 (33%) had stable disease or a partial response after the first cycle of treatment and proceeded to the second cycle. After the second cycle, 6 patients had stable disease, 4 had a partial response, and 1 had a complete response. Responses persisted for 21 to 106 weeks. CONCLUSIONS: Intratumoral plasmid DNA immunotherapy for head and neck cancer with Allovectin-7 is safe, and further investigations are planned in patients with less advanced disease, where it could potentially improve patient survival and reduce the need for radical high-morbidity treatments.

Analysis of the performance characteristics of the University of Washington Quality of Life instrument and its modification (UW-QOL-R).

BACKGROUND: During a 5-year period, we analyzed 3 patient subsets from the University of Washington Quality of Life (UW-QOL) Registry and published the results. In each instance, editorial review has raised legitimate concerns regarding the UW-QOL instrument that deserve public comment. We present our response to these criticisms. Since our original publication (1993), we have added domains to the original UW-QOL instrument. These additions reflected our concern that we might be missing important elements in the spectrum of disease-specific response to treatment. Using the data we have accumulated in the last 5 years, we present an analysis of the internal consistency of the UW-QOL. We have identified those domains that are responsive (or not responsive) to treatment effect and have revised the UW-QOL accordingly to create the UW-QOL-R, which is recommended for future use. DESIGN: The project began January 1, 1993, after approval by the UW Human Subjects Committee. Critical comments offered by external review were collated and responded to. Internal consistency was evaluated by interitem correlation matrix (Cronbach alpha) testing. SUBJECTS: All new patients presenting to the UW Medical Center (Seattle) with a diagnosis of head and neck cancer were asked to participate in a prospective analysis of QOL changes during and after treatment. INTERVENTION: Patients completed the pretreatment QOL questionnaire on the day of their initial workup. The format for the pretreatment test was an interviewer-supervised self-administered test; the subsequent tests were self-administered and were completed at 3, 6, 12, 24, and 36 months. Other data entered for each patient included site, stage, treatment, histologic classification, reconstruction, and current status. A QOL registrar was responsible for patient follow-up, data collection, and collation. All data were entered into the departmental relational database. RESULTS: Criticisms by external review included the following: "it is improper to call it [UW-QOL] a measure of quality of life"; "the summary scale is problematic because it implies that each of the subscales are weighted or 'valued' equally"; "some domain questions relate to surgery specific issues. while others are specific to radiation"; "we were confused by the scoring"; and "the UW-QOL index does not specifically address the psychological impact of the disease and its treatment." After evaluation of internal consistency, the UW-QOL was modified by removing 2 domains that correlated poorly with the others. This resulted in a 10-item instrument (UW-QOL-R) with an overall internal consistency score of 0.85. CONCLUSIONS: The UW-QOL can be effectively and accurately used to compare treatment effects in the management of head and neck cancer. With this revised instrument, the 10 items appear to measure the domains of overall QOL in a highly consistent and reliable fashion over time.

Basic fibroblast growth factor inhibits cell proliferation in cultured avian inner ear sensory epithelia.

Postembryonic production of inner ear hair cells occurs after insult in nonmammalian vertebrates. Recent studies suggest that the fibroblast family of growth factors may play a role in stimulating cell proliferation in mature inner ear sensory epithelium. Effects of acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2) were tested on progenitor cell division in cultured auditory and vestibular sensory epithelia taken from posthatch chickens. The effects of heparin, a glycosaminoglycan that often potentiates the effects of the FGFs, were also assessed. Tritiated-thymidine autoradiographic techniques and 5-bromo-2;-deoxyuridine (BrdU) immunocytochemistry were used to identify cells synthesizing DNA. The terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end-label (TUNEL) method was used to identify apoptotic cells. TUNEL and overall counts of sensory epithelial cell density were used to assess possible cytotoxic effects of the growth factors. FGF-2 inhibited DNA synthesis in vestibular and auditory sensory epithelia and was not cytotoxic at the concentrations employed. FGF-1 did not significantly alter sensory epithelial cell proliferation. Heparin by itself inhibited DNA synthesis in the vestibular sensory epithelia and failed to potentiate the effects of FGF-1 or FGF-2. Heparin was not cytotoxic at the concentrations employed. Results presented here suggest that FGF-2 may be involved in inhibiting cell proliferation or stimulating precursor cell differentiation in avian inner ear sensory epithelia.

Genetic and epigenetic changes in human epithelial cells immortalized by telomerase.

Exogenous expression of hTERT, the catalytic component of telomerase, is sufficient for the immortalization of human fibroblasts but insufficient for the immortalization of human foreskin keratinocytes (HFKs) and human mammary epithelial cells (HMECs). These latter cell types can overcome senescence by coexpression of hTERT and human papillomavirus (HPV) E7 or by expression of hTERT and loss of p16(INK4a) expression, indicating that the retinoblastoma (Rb) pathway, along with a telomere maintenance pathway, plays a role in determining the life span of epithelial cells. In this study, we further characterize hTERT-immortalized HFKs and human adenoid epithelial cells (HAKs) for genotypic and phenotypic alterations that are associated with immortalization. Of five hTERT-immortalized HFK and HAK cell lines examined, four exhibited repression of p16(INK4a) expression by promoter methylation or specific large-scale deletion of chromosome 9p, the location of p16(INK4a). Interestingly, one cell line exhibited complete down-regulation of expression of p14(ARF), with only slight down-regulation of expression of p16(INK4a). Yet, all of the immortal cells lines exhibited hyperphosphorylated Rb. Cytogenetic analysis revealed clonal chromosome aberrations in three of the five cell lines. All of the cell lines retained a growth block response with the expression of mutant ras. When grown on organotypic raft cultures, however, the hTERT-immortalized cells exhibited a maturation delay on terminal differentiation. Our results indicate that immortalization of epithelial cells may require both activation of telomerase and other genetic and/or epigenetic alterations that abrogate normal differentiation.

Quality of life in head and neck cancer.

Because treatments for patients with cancer of the head and neck can have major impact on physical, social, and psychological function, the collection of quality of life (QOL) data in this group of patients is critical for our specialty. The University of Washington Quality of Life data have been collected and analyzed on three subsets of cancer patients. Information learned from these patients is summarized and strategies for future projects are outlined.

Quality of life in patients with head and neck cancer: lessons learned from 549 prospectively evaluated patients.

OBJECTIVES: To summarize our quality-of-life (QOL) research findings for patients with head and neck cancer, to suggest areas for future productive QOL research, and to discuss how to undertake QOL studies in a cost-effective manner. DESIGN: Review of previously published analyses of advanced larynx cancer, advanced oropharynx cancer, and neck-dissection cases and current data from the complete set of patients. PATIENTS: From January 1, 1993, through December 31, 1998, data on 549 patients were entered in our head and neck database. Of these patients, 364 met additional criteria for histologic findings (squamous cell carcinoma) and the restriction of their cancer to 4 major anatomical sites (oral, oropharynx, hypopharynx, or larynx). Of these, 339 patients were more than 1 year beyond initial treatment. Complete baseline TNM staging and QOL data were obtained for 260 of these patients, of whom 210 presented with an untreated first primary tumor (index cases) to the University of Washington, Seattle. INTERVENTION: Pretreatment QOL was assessed with an interviewer-supervised self-administered questionnaire. Subsequent self-administered tests were completed at 3, 6, 12, 24, and 36 months. Other data collected on each patient included cancer site, stage, treatment, histologic findings, type of surgical reconstruction, and current disease and vital status. RESULTS/CONCLUSIONS: It is difficult to achieve "statistically significant" results in a single-institution setting. The "composite" QOL score may not be a sufficiently sensitive tool. Analysis of separate domains may be more effective.

Expression of Epstein-Barr virus latent membrane proteins leads to changes in keratinocyte cell adhesion.

Epstein-Barr virus (EBV) has 3 latent membrane proteins (LMPs)--LMP1, LMP2a, and LMP2b--which are expressed in nasopharyngeal carcinoma. Using keratinocyte cell lines expressing LMP2a and LMP2b and coexpressing LMP1/LMP2a, we grew organotypic raft cultures to analyze changes in morphology and expression of the cell adhesion molecule ICAM-1; alpha2, alpha3, alpha5, beta1, and alpha6beta4 integrins; laminin 5; E-cadherin; and desmoplakin. Cells expressing LMP2a or LMP2b were defective in their ability to mature and progress through normal squamous stratification when compared to the parental cell lines. Cells coexpressing LMP1/LMP2a additionally demonstrated "pseudoinvasion" into the raft dermal equivalent. There was a consistent and dramatic up-regulation in the suprabasal expression of laminin 5 and alpha6beta4 and beta1 integrins in the LMP-expressing cell lines. ICAM-1, not expressed in the control cell lines, was up-regulated in the LMP-expressing cell lines. Expression of alpha3 and alpha5 integrins was also up-regulated in the LMP-expressing cell lines, while alpha2 demonstrated a loss of the normal basal layer expression. E-cadherin and desmoplakin expression patterns were essentially unchanged. We conclude that LMP2a and LMP2b singly, and LMP1/LMP2a coexpressed, are capable of altering keratinocyte cell adhesion molecule expression consistent with nasopharyngeal carcinoma.

Quality of life after laryngectomy: are functional disabilities important?

BACKGROUND: The purpose of this study was to determine the functional disabilities and overall quality of life (QOL) of patients successfully treated (ie, without evidence of disease at two years) for laryngeal or hypopharyngeal cancer by a total laryngectomy. METHODS: The University of Washington QOL questionnaire was administered to 10 patients prior to laryngectomy, at one year postlaryngectomy, and at two years postlaryngectomy. RESULTS: Postlaryngectomy QOL scores were not significantly different from prelaryngectomy scores. In all QOL domains the severity of functional disability was not significantly correlated with its importance. Ninety percent of patients (9/10) reported that compared with one year prior to the diagnosis of cancer their general health was the same or better at two years postlaryngectomy. Seventy percent of patients (7/10) reported having a good to excellent overall QOL. CONCLUSIONS: Though the loss of voice is disabling, the functional limitations caused by a laryngectomy do not necessarily translate into a worse overall QOL. Future research is needed to determine whether the importance of individual QOL domains changes as patients adjust to the experience of having and surviving cancer.

Abnormal expression of the cystic fibrosis transmembrane regulator in chronic sinusitis in cystic fibrosis and non-cystic fibrosis patients.

Cystic fibrosis (CF) patients commonly suffer from chronic sinusitis. Mutations of a single gene, the cystic fibrosis transmembrane conductance regulator (CFTR) gene, have been associated with CF. Functional CFTR protein is localized to the apical cell membrane, while dysfunctional CFTR is commonly found in the cytoplasm. We undertook a preliminary immunocytochemical study of CFTR subcellular localization in CF and non-CF pediatric and adult patients using a newly developed murine monoclonal antibody, TAM. Immunostaining was evaluated for subcellular localization (cytoplasmic versus membranous) and for epithelial layer (basal versus luminal). Analysis of the predominant CFTR distribution patterns demonstrated significant differences in adult versus pediatric groups independent of whether the latter were CF or non-CF (p<.0001 and p<.008, respectively), and no significant difference between the 2 pediatric groups (p = .70). This suggests that the pathophysiology of pediatric sinusitis differs from that of adult sinusitis at the level of secretion production.

Primary T-cell lymphoma of the thyroid.

BACKGROUND: The routine use of immunocytochemical analysis has led to the recognition that many thyroid neoplasms previously diagnosed as anaplastic or small cell carcinomas are actually lymphomas of the thyroid. The great majority are B-cell lymphomas which can be associated with Hashimoto's thyroiditis. In spite of this, thyroid lymphomas are still not commonly recognized as a significant part of thyroid differential diagnosis. METHODS: A rare case of a primary T-cell lymphoma of the thyroid gland is presented along with general clinical history and physical findings which should make the practitioner suspicious of a thyroid lymphoma. The usefulness of radiology scans and fine-needle aspiration are discussed. RESULTS: Both prognosis and treatment options are very different for thyroid lymphomas and anaplastic carcinoma. CONCLUSIONS: Cyclophosphamide/adriamycin/vincristine/prednisolone chemotherapy/radiotherapy regimens have proven to be very effective for most thyroid lymphomas.

Thyroid cartilage fracture repair in rabbits: comparing healing with wire and miniplate fixation.

OBJECTIVES: Evaluate cartilaginous healing in rabbits in response to surgically created thyroid cartilage fractures. Compare healing between laryngeal fracture repair techniques. STUDY DESIGN: Animal model. MATERIALS AND METHODS: Laryngectomy specimens were analyzed at 10 weeks, following paired wire fixation (n = 7) and miniplate fixation (n = 7) of thyroid cartilage fractures. RESULTS: Cartilaginous unions were present in all seven of the miniplated repairs, while fibrous unions were present in six of the wired repairs. The measure of distraction at the fracture site was significantly greater in the wired repairs compared with the plated repairs (P = .005). Furthermore, in five of seven miniplated repairs no distraction at the healed fracture site was present. CONCLUSIONS: The results demonstrate the ease, tolerability, and superiority of the miniplate fixation technique for the thyroid cartilage fractures, based on a rabbit model.

Immunophenotype of ovarian cancer as predictor of clinical outcome: evaluation at primary surgery and second-look procedure.

OBJECTIVE: This study was undertaken to evaluate whether immunophenotyping of advanced epithelial ovarian cancer could predict response to initial chemotherapy and whether tumor immunophenotype changed after chemotherapy. STUDY DESIGN: Fifty-four patients with stage III and IV ovarian cancer, treated at the University of Washington Medical Center, had pathology specimens evaluated. A subset of 23 patients also had specimens from a secondary surgery evaluated. Using immunocytochemistry, tumors were immunostained for overexpression of c-erb-B-2, epidermal growth factor receptor (EGFR), p53, and expression of the Ki67-defined antigen (a marker of cellular proliferation), tumor necrosis factor alpha (TNFalpha), estrogen receptor (ER), progesterone receptor (PR), and P-glycoprotein (P170, a marker of multidrug resistance). Twenty-four patients had a good response to chemotherapy (defined as a negative, or microscopically positive second look), and 30 had a poor response (defined as grossly positive second look or progressive disease). RESULTS: Comparison of tumor markers from the initial and the secondary surgeries revealed that the only significant change was in the Ki67-defined cell proliferation rate, which showed a marked reduction in those with a good response to chemotherapy (P = 0.002). Comparison of tumor markers at initial surgery between good and poor responders revealed a correlation with p53 expression. Good responders were less likely to have p53 overexpression compared to poor responders, and this result approached significance (P = 0.058). Comparison of tumor markers at secondary surgery revealed a significant reduction in Ki67-defined cell proliferation rate in good responders compared to poor responders (P = 0.01). No significant differences were found between good and poor responders for the other tumor markers evaluated. CONCLUSIONS: The only tumor markers to predict for response to chemotherapy were p53 at initial surgery (P = 0.058) and Ki67 indices at secondary surgery (P = 0.001). Expression of steroid hormone receptors, TNFalpha, and P-glycoprotein and overexpression of c-erb-B-2 or EGFR are not associated with chemoresistance.

Macrophage and microglia-like cells in the avian inner ear.

Recent studies suggest that macrophages may influence early stages of the process of hair cell regeneration in lateral line neuromasts; numbers of macrophages were observed to increase prior to increases in hair cell progenitor proliferation, and macrophages have the potential to secrete mitogenic growth factors. We examined whether increases in the number of leukocytes present in the in vivo avian inner ear precede the proliferation of hair cell precursors following aminoglycoside insult. Bromodeoxyuridine (BrdU) immunohistochemistry was used to identify proliferating cells in chicken auditory and vestibular sensory receptor epithelia. LT40, an antibody to the avian homologue of common leukocyte antigen CD45, was used to label leukocytes within the receptor epithelia. Macrophages and, surprisingly, microglia-like cells are present in normal auditory and vestibular sensory epithelia. After hair cell loss caused by treatment with aminoglycosides, numbers of macrophage and microglia-like cells increase in the sensory epithelium. The increase in macrophage and microglia-like cell numbers precedes a significant increase in sensory epithelial cell proliferation. The results suggest that macrophage and microglia-like cells may play a role in releasing early signals for cell cycle progression in damaged inner ear sensory epithelium.

Quality of life of disease-free survivors of advanced (stage III or IV) oropharyngeal cancer.

BACKGROUND: This study assessed the quality of life (QOL) of patients with advanced oropharyngeal cancer (stage III or IV) who were disease-free at 1 year posttreatment. METHODS: Between 1993 and 1994, 13 consecutive cases were identified from the University of Washington QOL registry. Patients were divided into two groups, depending on treatment: surgical group, 6 patients treated with surgical resection and postoperative radiotherapy; and nonsurgical group, 7 patients treated with definitive radiotherapy. RESULTS: Composite pretreatment and posttreatment QOL scores were similar for the two treatment groups. Subset analysis of QOL domains revealed that both treatment groups generally reported a worsening of chewing and swallowing. A worsening of appearance and of speech was more frequently reported by the surgical group. Sixty-seven percent of the surgically treated patients reported pain relief, as opposed to only 29% of the nonsurgical group. CONCLUSION: Composite QOL-score sensitivity may be compromised by inverse changes in individual QOL domains. Treatment-specific QOL domains may be more sensitive measures of outcome.

Validation of the S-phase specificity of histone (H3) in situ hybridization in normal and malignant cells.

Several different methods of measuring proliferation indices have been developed, including measurements of cellular DNA content (flow cytometry), S-phase incorporation of thymidine analogues into DNA (e.g., tritiated thymidine and 5'-bromodeoxyuridine), and immunostaining of cell cycle-restricted proteins (e.g., Ki-67 antigen and PCNA). Theoretical and practical problems with each method have made it difficult to compare absolute proliferation rates among cells of different lineages and degrees of malignancy. More recently, in situ hybridization (ISH) for histone 3 (H3) mRNA has been introduced. We used a double labeling method for comparing H3 mRNA expression and S-phase incorporation of 5'-bromodeoxyuridine (BrdU) to determine if H3 mRNA expression was tightly associated with S-phase in a variety of malignant and nontransformed cell types. In addition, labeling results were compared in methacarn- and formalin-fixed tissues to extend the potential usefulness of H3 ISH, using a postfixation technique for the alcohol-fixed specimens. As expected for a cumulative marker, variation was noted in the percentage of the BrdU-positive cells double labeled with H3 ISH (53-89%), depending on cell type and length of BrdU incubation. In contrast, the percentage of the H3 ISH-positive cell population double labeled for BrdU was independent of the cell type of BrdU incubation time (mean 78%). Similarly, a consistent percentage of H3 ISH-positive cell populations was double labeled for BrdU in normal tissues (mean 97%). These findings support a well-conserved timing mechanism for H3 mRNA expression and DNA replication. We conclude that H3 ISH is an extremely accurate technique for assessment of S-phase cell proliferation indices.

Abnormalities of p53 and p110RB tumor suppressor gene expression in human soft tissue tumors: correlations with cell proliferation and tumor grade.

The authors tested the hypothesis that alterations in tumor suppressor gene expression play a role in tumorigenesis of human soft tissue tumors through alterations in control of cell proliferation. Using a set of 66 soft tissue tumors, including benign tumors and all three grades of sarcomas, expression of the p53 and p110RB tumor suppressor gene products were localized using sensitive immunocytochemistry techniques. The hypothesis that alterations in tumor suppressor gene expression was related to cell proliferation was tested by simultaneously demonstrating the expression of the proliferating cell nuclear antigen in methacarn-fixed, paraffin-embedded tissue sections of the same tumors. Twenty-two of 66 (33%) and 35 of 68 (53%) tumors demonstrated any degree of p53 overexpression or loss of p110RB, respectively. A strong correlation between increasing tumor grade and both p53 overexpression (P = 0.006) and loss of p110RB (P = 0.003) was found. Although there was a correlation between increasing proliferating cell nuclear antigen index and overexpression of p53 (P = 0.04), no correlations were found between cell proliferation indices and loss of p110RB (P = 0.19). Finally, there was a significant correlation between the presence of immunocytochemically detectable p53 overexpression and detectable p110RB loss (P = 0.02). These studies suggest that although alterations in p110RB may play a role in soft tissue sarcoma tumorigenesis and be related to p53 dysfunction, p110RB may act through mechanisms other than direct loss of cell proliferation control.

Cell cycle progression in gentamicin-damaged avian cochleas.

Hair cells, the sensory receptors of the auditory, vestibular, and lateral-line organs, may be damaged by a number of agents including aminoglycoside antibiotics and severe overstimulation. In the avian cochlea, lost hair cells can be replaced by regeneration. These new hair cells appear to be derived from a support cell precursor which is stimulated to divide by events associated with hair cell loss. Little is known about the timing and sequencing of events leading to new hair cell production. In this study cell cycle-associated events in the avian cochlea were analyzed at early and late time intervals following a single high dose of gentamicin. This single dose protocol has been shown to consistently result in extensive morphological damage and hair cell loss in the proximal region of the cochlea while sparing a morphologically undamaged distal cochlear region. This allowed for the differential analysis of the underlying support cell populations with respect to local hair cell loss. Three cell cycle associated markers were used to evaluate which cells entered and progressed through the cell cycle: statin, a G0 associated nuclear marker; proliferating cell nuclear antigen (PCNA), a G1, S and G2 associated marker; and 5-bromodeoxyuridine (BrdU), an S phase associated marker. Using these markers we found evidence for reversible changes in cell cycle status throughout the cochlea, while progression through S phase and mitosis was restricted to the region of the cochlea which sustained hair cell loss.

p53 and mdm-2 expression in malignant melanoma: an immunocytochemical study of expression of p53, mdm-2, and markers of cell proliferation in primary versus metastatic tumors.

Alterations in the function of p53, a tumor suppressor gene, have been postulated as a principal underlying mechanism involved in the loss of cell cycle control in human malignancies. Because p53 dysfunction is generally associated with protein overexpression, immunocytochemistry is a valuable technique for the analysis of p53's functional status. We tested the hypothesis that loss of p53 function is a critical event in the early development and progression of human malignant melanoma and can lead to alterations in cell proliferation. We performed an immunocytochemical study in archival fixed, embedded specimens that included 102 melanocytic lesions ranging from benign nevi to metastatic melanoma. In addition to p53, we assessed the p53-associated protein, mdm-2, and markers of cell cycle status (the MIB-1-defined cell proliferation marker; proliferating cell nuclear antigen; and statin, a 57-kDa nuclear protein expressed preferentially by G0 cells). Tumor expression of all nuclear proteins was scored in a semiquantitative fashion related to the fraction of positive tumor nuclei. The overall incidence of significant p53 overexpression was low (8% of primary and 14% of metastatic melanomas). Analysis demonstrated strong correlation between increasing p53 expression in primary versus metastatic lesions (chi 2 analysis, P = 0.001). Correlation was found between increased MIB-1-defined cell proliferation and p53 overexpression in primary melanomas (P = 0.02). Detectable mdm-2 expression was significantly correlated with p53 overexpression (P = 0.02). Comparison of statin and proliferating cell nuclear antigen indices demonstrated inverse correlation (chi 2 , P = 0.03) in the combined groups, but within the metastatic group there was a subset of cases strongly expressing the two markers.(ABSTRACT TRUNCATED AT 250 WORDS)