Lack of P-glycoprotein expression by low-dose fractionated radiation results from loss of nuclear factor-kappaB and NF-Y activation in oral carcinoma cells.

Multidrug resistance (MDR) is associated with the overproduction of the 170-kDa transmembrane protein P-glycoprotein (MDR1) caused by transcriptional activation. However, the activity of the MDR1 promoter in response to different doses of ionizing radiation has not been investigated. In this study, two squamous cell carcinoma oral cavity cell lines, T-167 and T-409, were exposed to either a standard clinical dose of 2 Gy or low-dose fractionated radiation therapy (LDFRT), delivered as 0.5 Gy in four fractions. MDR1 gene expression and degree of cell death were assessed. Clinically relevant 2-Gy dose of radiation resulted in increased expression of MDR1 by reverse transcription-PCR and luciferase reporter assays in both cell lines (T-167 and T-409), whereas LDFRT did not. LDFRT caused enhanced apoptosis when compared with the 2-Gy dose in T-167 and T-409 cells as assessed by terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) assays. Transcription of the MDR1 gene is regulated by numerous transcription factors, which include nuclear factor-kappaB (NF-kappaB), NF-Y, SP1, YB1, MEF1 (MDR1 promoter-enhancing factor 1), p53, and NF-R1. Interestingly, 2 Gy robustly induced both NF-kappaB and NF-Y in T-167 and T-409 cells, but did not show induction when exposed to LDFRT. Silencing the expression of the DNA binding subunit of NF-kappaB, p50, by small interfering RNA vector resulted in a decrease of MDR1 function by rhodamine 123 efflux assay in T167 cells exposed to 2 Gy. Together, these results provide evidence for the lack of induction of P-glycoprotein expression by LDFRT, which has important implications in combinatorial cancer therapy, including the use of LDFRT as an adjuvant for chemotherapy.

Gelatin sponge insertion into tympanostomy tubes: an in-vitro study to evaluate postoperative obstruction.

OBJECTIVE: To analyze the efficacy of gelatin sponge insertion into lumens of tympanostomy tubes to prevent obstruction in the presence of blood. STUDY DESIGN: In vitro model. METHODS: Absorbable gelatin sponge wicks were placed in the lumen of Ultrasil Collar Button ventilation tubes and Shepherd Grommet ventilation tubes. One half of each group was covered with blood, the other left untreated. Each tube was treated with ofloxacin solution three times daily for seven days. After treatment, the tubes were inspected. Reinspection was performed after brief suctioning. Numerical scores were given based on degree of obstruction. RESULTS: A statistically significant difference in degree of obstruction (P < 0.0001) was seen between all tubes with wicks alone versus those with blood added. After re-evaluation, there remained a statistically significant difference between tubes with wicks alone and tubes with wicks and blood (P < 0.0001). CONCLUSIONS: Gelatin sponge insertion does not prevent, and may in fact, enhance, obstruction of pressure equalization tube lumens in the presence of blood.

Incidence of thyroid carcinoma in fluorodeoxyglucose positron emission tomography-positive thyroid incidentalomas.

OBJECTIVE: Fluorodeoxyglucose (FDG) whole body positron emission tomography (PET) scan may show clinically occult second lesions. Such lesions in the thyroid are increasingly common. There are several recent reports of a high probability of malignancy in these lesions ranging from 14% to 63%. STUDY DESIGN AND SETTING: This is a retrospective review of 15,711 PET scans at a multi-disciplinary thyroid clinic at a tertiary care university medical center. Twenty-two patients were referred with thyroid PET "incidentalomas." The review included 18 FDG-PET scans, ultrasound guided fine needle aspiration biopsies, and thyroid surgery pathology. Aspiration cytology or pathology were the main outcome measures. RESULTS: Three patients had malignancy of the PET-positive thyroid lesions. Papillary thyroid micro carcinomas were detected in four of the specimens that showed a benign pathology of the dominant nodule. CONCLUSION: Our experience shows a 14% malignancy rate for the dominant (imaged) nodule and a total malignancy rate of 32% when the incidental micro carcinomas are included. Both of these rates are significantly lower than results published previously.

Analysis of unknown primary carcinomas metastatic to the neck: diagnosis, treatment, and outcomes.

Metastatic squamous cell carcinoma presenting in the neck from an unknown primary site represetns 2% to 6% of head and neck cancers. Optimal management of these cases remains controversial and continues to evolve with experience. We performed a retrospective analysis involving patients treated for unknown primary squamous cell carcinomas with metastases to cervical lymph nodes who presented to either the University of Kentucky or the Veterans Affairs Hospital of Lexington, Kentucky, from 1990 to 2000. Thirty-five out of 173 patients met inclusion criteria for carcinoma of unknown primary. The following data subsets were analyzed: age, gender, smoking and alcohol use, family history, diagnostic studies performed, radiation dose, surgical intervention, number and location of pathologic nodes, presence or absence of extracapsular extension, time between surgery and radiation, disease-specific and overall survival, response to treatment, emergence of a primary tumor, and duration of follow-up. Overall and disease-specific survivals were analyzed using, the Kaplan-Meier method and the log-rank test was used to assess differences in survival curves. The actuarial 5-year overall and disease-specific survival of all patients in this study was 54% and 63%, respectively. At 10 years, the overall survival declined to 37% with a disease-specific survival rate of 49%. The 5-year survival rates stratified by nodal stage were 80% for N1 patients, 64.7% for N2, 55.6% for N3, and 0% for any M disease. These rates declined to 60% for N1, 52.9% for N2, 11.1% for N3, and 0% for any M disease at 10 years (p<.0001). The presence of extracapsular spread, increased number of positive lymph nodes, and eventual discovery of a primary tumor did not significantly decrease survival in this series. The mean follow-up period for patients in this study was 54.8 months. We continue to refine our diagnostic and treatment strategies in this group of patients in an effort to improve long-term survival and reduce patient morbidity.

Low-dose fractionated radiation potentiates the effects of Paclitaxel in wild-type and mutant p53 head and neck tumor cell lines.

This study was designed to: (a) evaluate the induction of hyper-radiation sensitivity (HRS), a phenomenon observed at low doses of radiation (<1 Gy); (b) compare the potentiating effects of single dose radiation (2 Gy) versus the effect of low-dose fractionated radiation (LDFRT; <1 Gy) on Paclitaxel; and (c) understand the molecular mechanism of LDFRT-mediated chemo-potentiating effects, in wild-type p53 SCC-61 and p53 mutant SQ-20B head and neck squamous cell carcinoma cell lines. Both cell lines exhibited the HRS phenomenon at low radiation doses. Compared with SCC-61 cells, SQ-20B cells were resistant to radiation and Paclitaxel alone. A significant enhancement of radiation sensitization by Paclitaxel (0.5 or 1 nM) was observed in both cell lines. Chemo-potentiation of Paclitaxel by single 2-Gy radiation was observed in SCC-61 cells but not in SQ-20B cells. However, LDFRT (0.5 Gy in four fractions) significantly chemo-potentiated the effect of Paclitaxel in both cell lines. The cell cycle regulator p53 and its target genes p21(waf1/cip1) and BAX were induced in SCC-61 cells treated with 2 Gy, Paclitaxel, or in combination, but not in SQ-20B cells. These treatments elevated the antiapoptotic BCL-2 protein in SQ-20B cells but not in SCC-61 cells. Interestingly, LDFRT treatment in both cell lines with or without Paclitaxel down-regulated nuclear factor kappa B activity and BCL-2 protein expression and simultaneously up-regulated BAX protein. These findings strongly suggest that LDFRT (at these doses, HRS phenomenon is observed) can be used in combination with Paclitaxel to overcome the antiapoptotic effects of BCL-2 and nuclear factor kappa B.

Low dose fractionated radiation potentiates the effects of taxotere in nude mice xenografts of squamous cell carcinoma of head and neck.

This study evaluated the combined effect of Low Dose Fractionated Radiation (LDFRT) and Taxotere (TXT) therapy on the growth of SCCHN (squamous cell carcinoma of head and neck; SQ-20B, a p53 mutant SCCHN cell line) tumors in a nude mouse model to exploit the increased hyper radiation sensitivity (HRS) phenomenon present in G(2)/M cell cycle phase when induced by low doses of radiation that was demonstrated in in vitro settings. Seventy-eight animals were randomized into one control group and 5 treatment groups (treatments were administered weekly for six weeks). Tumor regression was observed in all the groups, however, tumor regression was not significant in 2 Gy or TXT or 2 Gy plus TXT treated groups when compared to control group. The tumor regression was significant in both the LDFRT group (p < 0.0043) and LDFRT + TXT group (p < 0.0006) when compared to other groups. A significantly prolonged tumor growth delay was observed in LDFRT group (p < 0.0081). Importantly, in combination of TXT and LDFRT, no tumor regrowth was observed in 12 out of 13 mice since LDFRT + TXT treatment caused a sustained regression of tumors for 9 weeks. Molecular analysis of resected tumor specimens demonstrated that Bax levels were elevated with concomitant increase in cytochrome c release to the cytosol of the treatment Group VI. These findings strongly suggest that LDFRT can be used in combination with TXT to potentiate the effects of drug on tumor regression through an apoptotic mode of death. Furthermore, the G(2)/M cell cycle arrest by TXT appears to be an important component of the enhanced apoptotic effect of TXT + LDFRT combined treatment.

MicroRNA expression profiles of head and neck squamous cell carcinoma with docetaxel-induced multidrug resistance.

BACKGROUND: The purpose of this study was to investigate the potential role of variable microRNA (miRNA) expression in the development of multidrug resistance (MDR) in head and neck cancer. METHODS: Head and neck squamous cell carcinoma cell lines UMSCC-1 and SQ20B were treated with docetaxel at increasing concentrations to develop resistant cell lines. Parental and resistant cells were treated with cisplatin, 5-fluorouracil, paclitaxel, methotrexate, and doxorubicin to confirm cross-resistance. The miRNA pattern of resistant cells was then compared with their parental cells. RESULTS: Docetaxel treatment successfully induced resistance primarily and induced multidrug cross-resistance. Resistant cells showed significant downregulation of miR-100, miR-130a, and miR-197 and upregulation in miR-101, miR-181b, miR-181d, and miR-195 expression when compared with their parent cells (p < .01). Real-time polymerase chain reaction (PCR) analysis confirmed statistically significant downregulation in miR-100 and miR-130a and upregulation in miR-181d expression (p < .001). CONCLUSION: Alterations in miRNA expression has direct relationship to MDR in head and neck cancer and may serve as biomolecular targets for reversal of MDR.

Novel neoadjuvant immunotherapy regimen safety and survival in head and neck squamous cell cancer.

BACKGROUND: Cellular immune suppression is observed in head and neck squamous cell cancer (HNSCC) and contributes to poor prognosis. Restoration of immune homeostasis may require primary cell-derived cytokines at physiologic doses. An immunotherapy regimen containing a biologic, with multiple-active cytokine components, and administered with cytoxan, zinc, and indomethacin was developed to modulate cellular immunity. METHODS: Study methods were designed to determine the safety and efficacy of a 21-day neoadjuvant immunotherapy regimen in a phase 2 trial that enrolled 27 therapy-naïve patients with stage II to IVa HNSCC. Methods included safety, clinical and radiologic tumor response, disease-free survival (DFS), overall survival (OS), and tumor lymphocytic infiltrate (LI) data collection. RESULTS: Acute toxicity was minimal. Patients completed neoadjuvant treatment without surgical delay. By independent radiographic review, 83% had stable disease during treatment. OS was 92%, 73%, and 69% at 12, 24, and 36 months, respectively. Histologic analysis suggested correlation between survival and tumor LI. CONCLUSION: Immunotherapy regimen was tolerated. Survival results are encouraging.

Implications of head and neck cancer treatment failure in the neck.

OBJECTIVE: Study the survival of patients with cervical lymphatic squamous cell carcinoma recurrence. STUDY DESIGN: Review of tumor registry database. SETTING: Academic health science center. SUBJECTS AND METHODS: Forty-seven isolated neck recurrence patients identified from 224 recurrences from a total of 1291 patients treated between 1998 and 2007. The main outcome measurements were neck lymph nodal recurrence, treatment-specific survival, and overall survival. RESULTS: A total of 47 patients had neck recurrence; 10 of the neck recurrence patients (21.3%) had regional disease (N+) at initial presentation. Median survival for patients with neck recurrence was 14.7 months (95% confidence interval [CI] 8.6-18.1 mo), and five-year survival for this group was five percent (95% CI 0%-30%). Neck dissection salvage therapy for neck recurrence resulted in the best survival. CONCLUSION: Neck dissection as a salvage therapy for neck recurrence resulted in the best survival, and there was no survival benefit in terms of whether a patient had a neck dissection or not as his or her initial therapy.

In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser.

The circulating tumor cell (CTC) count has been shown as a prognostic marker for metastasis development. However, its clinical utility for metastasis prevention remains unclear, because metastases may already be present at the time of initial diagnosis with existing assays. Their sensitivity ex vivo is limited by a small blood sample volume, whereas in vivo examination of larger blood volumes may be clinically restricted by the toxicity of labels used for targeting of CTCs. We introduce a method for in vivo photoacoustic blood cancer testing with a high-pulse-repetition-rate diode laser that, when applied to melanoma, is free of this limitation. It uses the overexpression of melanin clusters as intrinsic, spectrally-specific cancer markers and signal amplifiers, thus providing higher photoacoustic contrast of melanoma cells compared with a blood background. In tumor-bearing mouse models and melanoma-spiked human blood samples, we showed a sensitivity level of 1 CTC/mL with the potential to improve this sensitivity 10(3)-fold in humans in vivo, which is impossible with existing assays. Additional advances of this platform include decreased background signals from blood through changes in its oxygenation, osmolarity, and hematocrit within physiologic norms, assessment of CTCs in deep vessels, in vivo CTC enrichment, and photoacoustic-guided photothermal ablation of CTCs in the bloodstream. These advances make feasible the early diagnosis of melanoma during the initial parallel progression of primary tumor and CTCs, and laser blood purging using noninvasive or hemodialysis-like schematics for the prevention of metastasis.

Targeting human 8-oxoguanine DNA glycosylase (hOGG1) to mitochondria enhances cisplatin cytotoxicity in hepatoma cells.

Many chemoradiation therapies cause DNA damage through oxidative stress. An important cellular mechanism that protects cells against oxidative stress involves DNA repair. One of the primary DNA repair mechanisms for oxidative DNA damage is base excision repair (BER). BER involves the tightly coordinated function of four enzymes (glycosylase, apurinic/apyrimidinic endonuclease, polymerase and ligase), in which 8-oxoguanine DNA glycosylase 1 initiates the cycle. An imbalance in the production of any one of these enzymes may result in the generation of more DNA damage and increased cell killing. In this study, we targeted mitochondrial DNA to enhance cancer chemotherapy by over-expressing a human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene in the mitochondria of human hepatoma cells. Increased hOGG1 transgene expression was achieved at RNA, protein and enzyme activity levels. In parallel, we observed enhanced mitochondrial DNA damage, increased mitochondrial respiration rate, increased membrane potential and elevated free radical production. A greater proportion of the hOGG1-over-expressing hepatoma cells experienced apoptosis. Following exposure to a commonly used chemotherapeutic agent, cisplatin, cancer cells over-expressing hOGG1 displayed much shortened long-term survival when compared with control cells. Our results suggest that over-expression of hOGG1 in mitochondria may promote mitochondrial DNA damage by creating an imbalance in the BER pathway and sensitize cancer cells to cisplatin. These findings support further evaluation of hOGG1 over-expression strategies for cancer therapy.

Long-term results of hyperfractionated radiation and high-dose intraarterial cisplatin for unresectable oropharyngeal carcinoma.

BACKGROUND: In this report, the authors present the results from a study of patients with unresectable oropharyngeal squamous cell carcinomas who were treated on a protocol of hyperfractionated radiation and high-dose intraarterial cisplatin (HYPERRADPLAT) at the University of Kentucky. METHODS: The study was designed as a prospective, single-armed case series that was conducted in the setting of a single, academic, tertiary referral center. The patient cohort consisted of 24 previously untreated patients who were diagnosed with unresectable oropharyngeal carcinoma and were treated on the HYPERRADPLAT regimen, which included hyperfractionated external beam radiotherapy (1.2 grays [Gy] twice daily) was given for 5 weeks (60 Gy) followed by high-dose intraarterial cisplatin (150 mg/m2) and sodium thiosulfate. Shrinking "large-field" portals were started on Week 6 and finished on Week 7 with a cumulative dose of 76.8-81.6 Gy. The main outcome measures of the study were the primary and neck response rates, the 2-year and 5-year overall survival and disease-specific survival rates, and acute and late treatment morbidity. RESULTS: The median follow-up was 77 months. Complete response rates at the primary and regional lymph nodes were both 88%. The 2-year overall survival and disease-specific survival rates were 57% and 68%, respectively; whereas the 5-year overall survival and disease-specific survival rates were 33% and 42%, respectively. Two patients had Grade 4 mucosal toxicity, and no patient experienced neurologic or significant hematologic toxicities. Within 1 year of treatment, 58% of patients had used a feeding tube. CONCLUSIONS: The HYPERRADPLAT regimen produced excellent response rates and overall survival rates comparable to those achieved by patients who had unresectable oropharyngeal carcinomas. Tolerance of the therapy was good, and further studies using HYPERRADPLAT with induction therapy may improve outcomes further in this subset of patients with unfavorable disease.

Interval pathologic assessments in patients treated with concurrent hyperfractionated radiation and intraarterial cisplatin (HYPERRADPLAT).

BACKGROUND: To minimize surgical morbidity, surgery should be performed within 2 to 3 months of completion of radiation therapy with or without chemotherapy. Pathologic demonstration of cancer at this interval is commonly used to justify early surgical salvage of residual primary head and neck cancer. These assumptions regarding head and neck cancer in patients treated with concurrent hyperfractionated radiation therapy and intraarterial supradose cisplatin (HYPERRADPLAT) have never been evaluated. METHODS: Post-HYPERRADPLAT clinical and pathologic findings in 42 patients with stage III and IV head and neck cancer were compared with their disease outcomes. All patients underwent an interval analysis of response at 6 to 10 weeks after completion of therapy, 28 of these patients had biopsies of the primary tumor site performed. RESULTS: Clinical findings of cancer with pathologic confirmation up to 4 months after therapy can be associated with eventual complete response (CR). Pathologic CR's from deep incisional biopsies can be associated with recurrent disease within 2 months. Six HYPERRADPLAT-treated patients underwent interval surgical resection of primary disease, and only the four patients with cancer identified in the resection specimen died of recurrent disease. CONCLUSION: In patients treated with HYPERRADPLAT, interval clinical and pathologic assessments may be misleading. Only observation of progressive disease is an accurate predictor of local failure. New evaluation techniques such as metabolic imaging and molecular analysis warrant exploration as tools for interval cancer evaluations.