Joseph Tung-Chien Chang

Prognostic significance of EGFR and Her-2 in oral cavity cancer in betel quid prevalent area cancer prognosis

Although several studies have found overexpression of epidermal growth factor receptor (EGFR) proteins EGFR and Her-2 in head and neck cancers, the clinical relevance of the finding varies. We examined the expression and clinical association of these molecules with oral squamous cell carcinoma in an area where betel chewing is prevalent. EGFR and Her-2 proteins were measured in 59 paired (grossly normal and cancer) tissues by an enzyme immunoassy method. The cutoff value for gene overexpression was defined as the level of mean expression in normal tissue plus two s.d. A total of 59% of the patients consumed alcohol, 90% smoked tobacco, and 90% chewed betel quid. Of the patients assayed, 34 (58%) and 24 (41%) had EGFR and Her-2 overexpression, with average 3.5- and 1.5-fold elevations. EGFR overexpression has been shown to be statistically associated with T stage, N stage, overall TMN stage, primary tumour depth, lymph node extra-capsular spread, and poor survival. Her-2 overexpression, however, did not demonstrate a similar association with clinicopathological parameters or therapeutic outcome. On multivariant analysis, EGFR overexpression (P=0.041) and N stage (P=0.024) were the only independent factors for overall survival. These results indicate that the molecular targeting therapy to EGFR may be a treatment for oral cavity cancer in the betel quid-chewing prevalent area.

DSG3 is overexpressed in head neck cancer and is a potential molecular target for inhibition of oncogenesis

To identify genes that could potentially serve as molecular therapeutic markers for human head and neck cancer (HNC), we employed differential display analysis to compare the gene expression profiles between HNC and histopathologically normal epithelial tissues. Using reverse transcription–polymerase chain reaction and Western blot analysis, desmoglein 3 (DSG3) was identified as being differentially expressed at both the RNA and protein levels. Of 56 patients assayed, 34 (61%) had overexpression of DSG3, which correlated statistically with T stage (P=0.009), N stage (P=0.047), overall stage (P=0.011), tumor depth (P=0.009) and extracapsular spread in lymph nodes (P=0.044), suggesting that DSG3 participates in carcinogenesis of HNC. Consistent with the clinical findings, inhibition of DSG3 by RNA interference (RNAi) significantly reduced cell growth and colony formation to 57–21% in three HNC cell lines. Use of an in vitro wound healing and Matrigel invasion assays, we found that cell migration and invasive ability were also inhibited to 30–48% in three cell lines tested. An in vivo xenograft study showed that administration of DSG3-RNAi plasmid significantly inhibited tumor growth for 2 months in BALB/C nude mice. In conclusion, DSG3 is identified overexpressed in HNC, with the degree of overexpression associated with clinicopathologic features of the tumor. Inhibition of DSG3 significantly suppresses carcinogenic potential in cellular and in vivo animal studies. These findings suggest that DSG3 is a potential molecular target in the development of adjuvant therapy for HNC.

hTERT phosphorylation by PKC is essential for telomerase holoprotein integrity and enzyme activity in head neck cancer cells

Telomerase activity is suppressed in normal somatic tissues but is activated in most cancer cells. We have previously found that all six telomerase subunit proteins, including hTERT and hsp90 are needed for full enzyme activity. Telomerase activity has been reported to be upregulated by protein kinase C (PKC), but the mechanism is not clear. In this study, we examined how PKC regulates telomerase activity in head and neck cancer cells. PKC inhibitor, bisindolylmaleimide I (BIS), inhibited telomerase activity but had no effect on the expressions of telomerase core subunits. RNA interference (RNAi) and in vitro phosphorylation studies revealed that PKC isoforms α, β, δ, ɛ, ζ specifically involved in telomerase regulation, and the phosphorylation target was on hTERT. Treatment with the hsp-90 inhibitor novobiocin dissociated hsp90 and hTERT as revealed by immunoprecipitation and immunoblot analysis and reduced telomerase activity. Treatment with the PKC activator SC-10 restored the association of hsp90 and hTERT and reactivate telomerase, suggesting that hTERT phosphorylation by PKC is essential for telomerase holoenzyme integrity and function. Analysis on clinical normal and tumour tissues reveal that the expressions of PKC α, β, δ, ɛ, ζ were higher in the tumour tissues, correlated with telomerase activity. Disruption of PKC phosphorylation by BIS significantly increased chemosensitivity to cisplatin. In conclusion, PKC isoenzymes α, β, δ, ɛ, ζ regulate telomerase activity in head and neck cancer cells by phosphorylating hTERT. This phosphorylation is essential for telomerase holoenzyme assembly, leading to telomerase activation and oncogenesis. Manipulation of telomerase activity by PKC inhibitors is worth exploring as an adjuvant therapeutic approach.

Prediction of supraclavicular lymph node metastasis in breast carcinoma

PURPOSE Supraclavicular lymph node metastasis in breast cancer patients has a poor prognosis, and aggressive local treatment has usually resulted in severe morbidity. The purpose of this study was to select high-risk neck metastasis patients for prophylactic radiotherapy. METHODS Between 1990 and 1998, 2658 consecutive invasive breast cancer patients underwent surgery and adjuvant therapy in the hospital. The median age was 47 years (range 22-92). The median follow-up period was 39 months. The following factors were analyzed: age, tumor size, tumor location, histologic type, histologic grade, estrogen and progesterone receptor status, DNA flow cytometry study results, number of positive axillary lymph nodes, use of chemotherapy, radiotherapy, and/or hormonal therapy, and level of involved axillary nodes. RESULTS Of the 2658 patients, 113 (4.3%) developed supraclavicular lymph node metastasis during this period. Young age (< or =40 years), tumor size >3 cm, high histologic grade, angiolymphatic invasion, negative estrogen receptor status, synthetic phase fraction >4%, >4 positive nodes, and level II or III involved nodes were all significant for predicting neck metastasis in the univariate analysis. Three predictive factors were significant after multivariate analysis: high histologic grade, >4 positive nodes, and axillary level II or III involved nodes. In patients with axillary level I involved nodes and < or =4 positive nodes, the incidence was 4.4%. If axillary level III was involved, the rate of supraclavicular lymph node metastasis was 15.1%. CONCLUSION The incidence of supraclavicular lymph node metastasis was higher in the groups with >4 positive nodes and in those with axillary level II or III involved nodes. Selective use of comprehensive radiotherapy for these high-risk patients will achieve good locoregional control.

Highly potent and specific siRNAs against E6 or E7 genes of HPV16- or HPV18-infected cervical cancers

Infection with high-risk types (type 16 or type 18) of human papillomaviruses (HPVs) increases a patients risk of cervical cancer. Given the importance of the cervix and the severe side effects resulting from traditional cancer therapies, this study aimed to achieve targeted inhibition of viral oncogenes in tumor cells using small interfering RNAs (siRNA). To accomplish this, we developed nine siRNAs against either the E6 or E7 genes of HPV-16 or HPV-18 in several combinations, yielding siRNAs targeting 16E6, 16E7, 18E6 and 18E7. We measured the effectiveness of the siRNAs by examining E6 or E7 mRNA expression after transfection of the siRNAs into HPV-positive CaSki (HPV-16) or HeLa (HPV-18) cell lines. We found that the HPV-siRNAs significantly reduced cell growth and colony formation in both cell lines. Flow cytometry analysis revealed a significant increase in apoptosis. The siRNAs had no effect on cell growth, colony formation or apoptosis in HPV-negative C33A cells, demonstrating a lack of off-target effects. In addition, an in vivo xenograft study showed that intra-tumoral injection of the siRNAs reduced tumor growth in BALB/c nude mice. In conclusion, we have developed highly specific and potent HPV-siRNAs that successfully suppress tumor growth and induce apoptosis in HPV-positive cervical cancer cells. siRNA treatment has potential for further development as an adjuvant therapy for cervical cancer.

Linear accelerator-based radiosurgery in the management of skull base meningiomas.

From May 1994 to December 1999, 43 patients with meningiomas in the base of the skull underwent linear accelerator (LINAC)-based stereotactic radiosurgery (SRS) at Chung Gung Memorial Hospital. SRS was performed as a primary treatment in 14 patients, and after resection in 29 patients. The mean tumor volume was 5.68 cc, and the mean target surface dose was 16 Gy, delivered with an average of three isocenters. The median follow-up was 74.5 months. The 7-year local control rate and 7-year overall survival rate were 89.7% and 80.2%, respectively. The 7-year local control was 100% and 84.4% in SRS alone group and surgical excision with SRS group (p = 0.21), respectively. A stationary tumor following SRS was seen in 23 (53.5%) patients, partial shrinkage was seen in 16 (37.2%) patients, and complete disappearance in one patient was seen (2.3%). Furthermore, disease progression was noted in three (7%) patients, one of whom died of disease. The median time to tumor response was 15.4 months (range 5.8–52.8 months). Cases remained stable or had improved neurological statuses without any deterioration in 37.9% and 78.7% of the groups treated with surgery and SRS and SRS alone, retrospectively. In summary, LINAC-based SRS is an effective and safe modality of treating unresectable or partially resected meningiomas in the base of the skull. For tumors with diameters of 3 cm or less, particularly in patients without or with minimal clinical neurological symptom, SRS alone can provide a good local control without causing cranial neuropathy.

Unmet supportive care needs and characteristics of family caregivers of patients with oral cancer after surgery

The aim of this study was to identify factors associated with unmet supportive care needs in family caregivers of patients with oral cancer after surgery.

Grp78 as a therapeutic target for refractory head–neck cancer with CD24 − CD44 + stemness phenotype

Cancer stem cells are refractory to conventional therapy, which result to cancer metastasis and chemo-radioresistance. Grp78 is known to have important roles in cytoprotection and tumorigenesis in several cancers. We therefore examined whether Grp78 can serve as a therapeutic target for refractory stemness phenotype of head and neck cancer (HNC). Six HNC cell lines were used. Fluorescence-activated cell sorting (FACS) analysis was used to sort CD24−CD44+ and Grp78+ cells. The small interfering RNA (siRNA) knockdown and cDNA transfection were applied to examine the effects of Grp78 on cellular function. Western blot and confocol microscopy were used to determine the effects of downstream protein expressions. Xenografted mouse tumors and immunohistochemistry were used to validate the results. We found that Grp78 regulated the conversion of CD24−CD44+ cells, a characteristic of HNC stem cells. The CD24−CD44+Grp78+ cells showed superior chemo-radioresistance and invasion ability compared with CD24−CD44+, Grp78+ or the parental cells. Silencing Grp78 increased chemo-radiosensitivity, inhibited cell invasion, reverse epithelial–mesenchymal transition, suppressed cancer stemness, withdrew CD24−CD44+ cell conversion and induced differentiated phenotype. Study in xenografted mice further showed that CD24−CD44+Grp78+ cells exhibited highest tumorigenesis, compared with CD24−CD44+ CD24+CD44+ or the parental cells. Grp78 knockdown dramatically restrained tumor growth along with the inhibition of stem cell regulatory proteins Oct-4 and Slug. Grp78 may serve as a molecular target that can be further developed for eradication of refractory HNC with stemness phenotype.

Telomerase activity is frequently found in metaplastic and malignant human nasopharyngeal tissues.

Telomerase is a specialized ribonucleoprotein polymerase that directs the synthesis of telomere repeats at chromosome ends. Accumulating evidence has indicated that telomerase is stringently repressed in normal human somatic tissues but reactivated in cancers and immortal cells, suggesting that reactivation of telomerase plays an important role in carcinogenesis. In this study, the status of telomerase activity in diseased human nasopharyngeal lesions was determined by the telomeric repeat amplification protocol (TRAP). Fifty-four patients participated including 17 inflammation or hyperplasia, eight with squamous metaplasia, and 29 with different stages of carcinomas. Telomerase activity was detected in 1 of 17 (5.9%) inflammatory or lymphoid hyperplastic tissues, 3 of 8 (37.5%) squamous metaplastic, and 25 of 29 (86.2%) carcinoma tissues. The differences in telomerase expression in these groups is statistically significant (P< 0.001). Levels of telomerase activity correlated with tumour stage (P = 0.024). These results suggest that telomerase reactivation plays a role in the carcinogenesis of nasopharyngeal cancer. Since telomerase activity is found in the majority of nasopharyngeal cancers and a subset of metaplasia, this enzyme may be served as a reference to monitoring the status of abnormal nasopharyngeal tissues.

Orofacial pain and predictors in oral squamous cell carcinoma patients receiving treatment

Surgical and radiation therapy for oral squamous cell carcinoma (OSCC) may generate orofacial pain. The aims of this study were to (1) characterize the pain experienced by people with orofacial pain, and (2) determine the factors associated with changes in orofacial pain in OSCC patients during the postoperative and post-radiation therapy periods. The study had a prospective longitudinal design with consecutive sampling. Seventy-two eligible patients were recruited from the outpatient department of otolaryngology, head and neck cancer, and radiation therapy of a medical center in northern Taiwan. A set of questionnaires was used for patient assessment, including the University of California San Francisco Oral Cancer Pain Questionnaire, Symptom Severity Scale, Hospital Anxiety and Depression Scale, and Karnofskys Performance Status Index. Patients were assessed at two time points: 1 month after surgery (T1) and 1 month after completion of radiation treatment (T2). The findings showed that (1) patients reported moderate orofacial pain at both time points; (2) orofacial pain, oral function-related symptoms, and psychological distress were significantly higher at T1 than at T2; and (3) older age, eating difficulty, speech difficulty, and depression were significant predictors of orofacial pain. Oral rehabilitation and relaxation training may reduce orofacial pain in this patient population.