Quantitative assay of telomerase activity in head and neck squamous cell carcinoma and other tissues

OBJECTIVES: To confirm the applicability and use of a new technique to detect and quantify telomerase activity of specimens from head and neck malignant neoplasms and to explore whether the levels of telomerase activity can be a useful marker for cancer risk assessment in head and neck malignant neoplasms. DESIGN: Ninety-six specimens from 39 patients with head and neck malignant neoplasms were obtained. The specimens included 39 from patients with primary tumors (25 with head and neck squamous cell carcinoma and 14 with others), 10 from patients with neck metastases, 10 from patients with dysplasias, and 37 from patients with normal tissue. HeLa cell lines were used as positive control samples. MAIN OUTCOME MEASURE: The levels of telomerase activity were determined using a liquid scintillation counter. RESULTS: The new method has a high rate of outcome reproducibility. The intrabatch and extrabatch variations were 15.6% and 16.4%, respectively. The linear relationship was good between the telomerase activity and the value within 700 radioactive cpm (rcpm) to approximately 7000 rcpm. The levels of telomerase activity determined by radioactive count were more than 1000 rcpm in 42 of the 49 malignant specimens and much more than that in the normal tissues, with the exception of 3 specimens. The levels of telomerase activity in normal tissues were less than 1000 rcpm in every sample and less than that in the malignant neoplasm samples, with the exception of 1 specimen (P < .000). Higher levels of telomerase activity in 2 of 10 tissues from patients who had dysplasias were detected (2 specimens from patients who had severe dysplasia). The differences in the levels of telomerase activity between the head and neck squamous cell carcinomas and the other tumors were not statistically significant (P > .05). CONCLUSIONS: Detection of telomerase activity in head and neck malignant neoplasms can be a useful marker for the assessment of cancer. Telomerase reactivation may play an important role in tumorigenesis in head and neck squamous cell carcinoma. The quantification of telomerase activity may have clinical diagnostic value for head and neck malignant neoplasms.     

Motility-related proteins as markers for head and neck squamous cell cancer

HYPOTHESIS: Increased cell motility is a hallmark of cancer cells. Proteins involved in cell motility may be used as molecular markers to characterize the malignant potential of tumors. METHODS: Molecular biology and immunohistochemistry techniques were used to investigate the expression of a selected panel of motility-related proteins (Rho A, Rac 2, Cdc42, PI3K, 2E4, and Arp2) in normal, premalignant, and squamous cell cancer cell lines of human head and neck origin. To assess the clinical potential of these proteins as molecular markers for cancer, immunohistochemistry was performed on paraffin-fixed head and neck cancer specimens (n = 15). RESULTS: All six motility-associated proteins were overexpressed in the premalignant and squamous cell cancer cell lines relative to normal keratinocytes. Immunohistochemistry with Rho A and Rac 2 showed increased staining in areas of cancer but not in normal tissue. CONCLUSION: Proteins involved in cell motility can be used as markers for head and neck squamous cell carcinoma. The head and neck cell lines used in this study may be used as a model to further investigate cell motility. Molecular markers of motility could have a significant impact on the diagnosis and staging of cancers originating from differentiated non-motile cells.     

Urokinase-type plasminogen activator expression and proliferation stimulation in head and neck squamous cell carcinoma in vitro and in situ

BACKGROUND: Stimulation of proliferative activity by urokinase-type plasminogen activator (uPA) has been demonstrated in vitro for cultured primary and carcinoma cells. OBJECTIVE: To examine the effect of uPA stimulation on cultured squamous cell carcinoma cell lines of the head and neck in vitro and to compare the results with the situation in tumor tissue specimens. DESIGN: The uPA-mediated growth stimulation of 2 head and neck squamous cell carcinoma cell lines after suppression of endogenous uPA production was monitored by measuring (3)H-thymidine uptake into cellular DNA. Alternatively, applications of antibodies against the uPA-binding domain of the urokinase receptor were used to suppress autostimulation. To analyze the situation in situ we performed Western blot and zymographic studies on tissue homogenates of 25 squamous cell carcinoma specimens. We tested the expression of proliferating cell nuclear antigen (PCNA), a marker for proliferative activity, and uPA in tissue lysates and correlated uPA and PCNA expression by regression analysis. RESULTS: High-molecular-weight urokinase had a proliferation stimulative effect on both cell lines in vitro. The uPA autostimulation was decreased by blocking the uPA-binding domain of urokinase receptor with antibodies. Regression analysis of zymographic and Western blot data of tumor tissue lysates revealed no significant coherency between PCNA and uPA expression. Immunohistochemical stainings frequently showed different sublocalization of uPA and PCNA within tumors. CONCLUSION: In vitro uPA-mediated growth stimulation is not necessarily transferable to the in situ situation.     

Modulation of human leukocyte antigen class I expression by gamma interferon in head and neck cancer cell lines

One possible mechanism explaining the action of interferon (IFN) on squamous cell carcinoma (SqCC) of the head and neck is the modulation of major histocompatibility antigen expression on tumor cells. We tested the ability of gamma interferon (gamma-IFN) to modulate major histocompatibility class I antigens and beta 2-microglobulin (beta 2-M) on two carcinoma cell lines derived from SqCC of the head and neck. Major histocompatibility class I antigens and beta 2-M were detected using a two-step immunochemical stain; antigen expression was quantified using flow cytometry. gamma-IFN increased constitutive antigen expression by as much as five times on both cell lines. Maximum modulation was seen within 72 hours of exposure to gamma-IFN at clinically attainable doses (10 U/mL to 100 U/mL). The presence of gamma-IFN in cell cultures was necessary for continued modulation of surface antigens. These findings suggest a possible mechanism of action and encourage further clinical trials with gamma-IFN.     

Oncofetal antigen expression in head and neck carcinoma.

Oncofetal antigens (OFA) are proteins expressed during periods of embryonal/fetal development and on malignant cells. Previous investigations detected the presence of phase-specific 44- and 200-kd OFA, using a monoclonal antibody (MoAb 115). This study will determine if primary head and neck squamous cell carcinomas express the 44-kd OFA, and will assess the possible role of OFA in oncogenesis. Fifteen primary human head and neck squamous cell carcinomas (HNSCC) and six HNSCC cell lines were tested for OFA expression. Thirteen primary cancers and three cell lines demonstrated various degrees of OFA positivity. Oncofetal antigens are proposed to represent a proto-oncogene active during fetal development and malignant growth in head and neck squamous cell carcinomas.     

Epidermal growth factor receptors and oestrogen receptors in the head and neck

We report a pilot study of the levels of epidermal growth factor receptors (EGFR) in normal and neoplastic tissues of the head and neck. Specimens from 20 consecutive patients undergoing major head and neck surgery showed that squamous cell carcinomas had detectable but widely varying levels of EGFR. EGFR expression was greater in normal salivary gland tissue than in benign salivary neoplasms. Oestrogen receptors were not detected in clinically significant amounts in any of the tumours assayed.     

An immunohistochemical analysis of p53 protein expression in pre-malignant and malignant tissues of the oral cavity

This study looks at p53 protein expression in dysplastic and malignant lesions of the oral cavity using an immunohistochemical staining technique. Archival biopsy specimens of oral dysplasia of squamous cell carcinoma from 64 patients were analysed immunohistochemically. Sections from 90 oral biopsy specimens were examined in all. Positive immunohistochemical detection of the p53 protein, demonstrated by brown nuclear staining, was detectable in over 80% of mild, moderate and severe dysplastic tissues as well as carcinoma-in-situ and squamous carcinoma specimens. We concluded that p53 protein expression occurs frequently in both malignant and dysplastic lesions of the oral cavity, suggesting that abnormally detectable p53 protein is present at the very early stages of development of oral squamous carcinoma. Oral cancer may provide a good model for the study of multistage tumorigenesis in head and neck cancer as the lesions are frequently detected at the pre-invasive stage and are accessible to biopsy     

Is glutathione-S-transferase-pi expression a reliable predictor of chemoradiation response in cancer of the head and neck?

PURPOSE: Concurrent radiation and chemotherapy is being evaluated as an alternative treatment to surgery for patients with advanced squamous cell carcinoma of the head and neck, because organ preservation maybe possible without compromising survival. However, the response to concurrent chemoradiation treatment varies from patient to patient, and there is currently no available molecular predictor of response for this particular treatment modality. There is some evidence to indicate that glutathione S-transferase-pi (GST-pi), which is one of the drug detoxifying enzymes, may decrease the effectiveness of platinum-based chemotherapy in the treatment of a variety of tumor types. This study was performed to investigate whether GST-pi expression was correlated with response to concurrent chemotherapy and radiotherapy in patients with advanced squamous cell carcinoma of the head and neck. MATERIALS AND METHODS: Diagnostic biopsy specimens of 36 patients who underwent concurrent chemoradiotherapy for the treatment of advanced squamous cell carcinoma of the head and neck were examined for GST-pi expression by using immunohistochemistry with polyclonal antihuman GST-pi antibodies. GST-pi expression scores were compared among responders and nonresponders. RESULTS: Although the staining rate with antiGST-pi was slightly lower in the responder group in comparison with the nonresponders (82% vs 100%), the difference was not statistically significant. CONCLUSION: GST-pi expression is unlikely to be a valuable predictor of response to concurrent chemotherapy and radiation treatment in patients with advanced squamous cell carcinoma of the head and neck.     

Tumor deposition of laminin-5 and the relationship with perineural invasion.

OBJECTIVES/HYPOTHESIS: Perineural invasion (PNI) is increasingly being recognized as an important indicator of aggressiveness in head and neck squamous cell carcinoma. The mechanisms of PNI are poorly understood. Laminin-5, an important basement membrane constituent, has been shown to be essential in head and neck squamous cell carcinoma invasion and motility. We hypothesized that tumors exhibiting increased expression of laminin-5 are more likely to be neurotropic. STUDY DESIGN: Analysis of archived surgical specimens with and without PNI for presence and intensity of laminin-5 tumor staining. METHODS: Immunohistochemistry of archived head and neck squamous cell carcinoma specimens with known PNI was performed with anti-laminin-5 antibodies and appropriate positive and negative control specimens. The staining patterns were characterized as follows: A, few to no tumor cells positive; B, some peripheral cells positive; C, all peripheral cells positive; and D, almost all tumor cells positive. Statistical analysis was by chi2 analysis. RESULTS: Forty-six PNI-positive and 18 PNI-negative specimens were analyzed. The staining distribution for the PNI-positive specimens was as follows: 2% for A, 41% for B, 46% for C, and 11% for D. For tumors without PNI, the distribution was 28% for A, 50% for B, 22% for C, and 0% for D (P = .005). In PNI-positive tumors, no significant difference in staining was seen between areas with and without PNI. CONCLUSIONS: We found a significant correlation between laminin-5 staining and the presence of PNI in head and neck squamous cell carcinoma. Expression of laminin-5 by tumors is, possibly, an important step in the process of PNI. These preliminary findings support the concept that deposition of basement membrane constituents are required in the multistep process of nerve invasion.     

Integrins in head and neck squamous cell carcinoma invasion

OBJECTIVE: To relate the invasive properties of different squamous cell cancer cell lines to the function and expression of the integrins. STUDY DESIGN: A series of in vitro and in vivo experiments were designed to assess and compare integrin expression and function in two different head and neck squamous cell carcinoma cell lines. METHODS: Invasive properties of two squamous cell carcinoma cell lines (UM-SCC-1 and JHU-022-SCC) were assessed using an in vitro artificial matrix assay as well as an in vivo system with orthotopically implanted tumor cells in mice. Whole cell and surface expression levels of integrin subunits (alpha2, alpha3, alpha5, alpha6, beta1, and beta4) were determined for each cell line using Western blot analysis and flow cytometry. We compared the ability of JHU-022-SCC and UM-SCC-1 cells to bind the extracellular matrix elements collagen IV, fibronectin, laminin 5, and laminin10 using an in vitro adhesion assay. Contributions of the different integrins to the adhesive properties were determined by selective antibody blocking of different subunits. RESULTS: The UM-SCC-1 cell line is 50% more invasive in vitro and displays a greater propensity for perineural and lymphatic invasion in vivo. The UM-SCC-1 cells exhibited greater adherence to fibronectin than JHU-022-SCC cells. Alpha6 and beta4 expression is approximately twofold greater in the JHU-022-SCC cells. Alpha2, alpha3, and beta1 expression appears to be upregulated in UM-SCC-1 cells. CONCLUSION: The UM-SCC-1 carcinoma cells are more invasive than JHU-022-SCC cells and may be related to differential expression of the integrins alpha6beta4, alpha3beta1, and alpha2beta1.     

Management implications of evaluating the N2 and N3 neck after organ preservation therapy

OBJECTIVES/HYPOTHESIS: To determine if metastatic squamous cell carcinoma with proliferative potential persists in N2 and N3 necks after conventional radiation. STUDY DESIGN: Retrospective case series. MATERIALS AND METHODS: We identified 17 patients from our head and neck tumor database who underwent organ-preserving radiotherapy for primary aerodigestive squamous cell cancer and N2-3 regional metastasis. Archival tissue from these 17 neck specimens was evaluated for routine histopathologic evidence of tumor, as well as immunohistochemically for cytokeratin and Ki-67 activity. An assay for apoptosis was also performed on 10 of the specimens. RESULTS: Routine H&E evaluation suggested metastatic cancer in 11 of 17 irradiated neck specimens. Cytokeratin immunostaining confirmed squamous cell carcinoma in these 11 necks as well as 1 additional specimen that had tested H&E negative. Ki-67 staining demonstrated proliferating tumor in 3 of 17 necks. The apoptosis assay confirmed regions of apoptosis in all of the specimens analyzed. CONCLUSIONS: The discovery of proliferating cancer cells in 3 of 17 irradiated specimens (18%) supports the practice of planned neck dissection after primary radiotherapy for patients with pretherapeutic N2+ metastatic disease.     

Fine-needle aspiration in squamous cell carcinoma of the head and neck

Cytological examination of fine-needle aspiration (FNA) specimens from patients with known or suspected squamous cell carcinoma of the head and neck at the University of Virginia, Charlottesville, between 1979 and 1982 was reviewed. Of 229 aspirates, 187 were of cervical or submandibular sites and 42 were of oral cavity sites. The accuracy of cytological diagnoses was determined by histological comparison and clinical follow-up. There were no false-positive aspirates (specificity, 100%) and one false-negative aspirate (sensitivity, 99%). Twenty-one (9%) contained insufficient cellular material for adequate cytological diagnosis. Seventy-eight percent of histologically confirmed aspirations were malignant. Proper use requires close communication between an experienced cytologist and a head and neck surgeon knowledgeable in the indications and limitations of the technique. This study demonstrates that FNA is a highly accurate, safe, predictive, and valuable technique in the management of squamous cell carcinoma of the head and neck.     

头颈鳞癌血管生成与颈淋巴结转移相关性研究

目的 :探讨头颈鳞癌血管生成与其颈淋巴结转移的关系以及血管内皮生长因子(VEGF)在头颈鳞癌血管生成中的作用。方法 :应用免疫组化SABC法检测 5 8例头颈鳞癌组织中微血管密度 (IMVD)及VEGF的表达。结果 :5 8例头颈鳞癌组织中IMVD为 2 3.93± 8.77,肿瘤分化程度 ,高与中、高与低间 ,IMVD差异有显著性意义 (均P <0 .0 5 ) ;中与低间 ,差异无显著性意义 (P >0 .0 5 )。颈淋巴结转移组IMVD(2 7.92± 9.11)明显高于非转移组 (2 0 .6 9± 7.0 8) ,其差异有显著性意义 (P <0 .0 1)。癌组织中VEGF表达与瘤内IMVD呈正相关 (rs=0 .4 87,P <0 .0 1)。结论 :瘤内IMVD可作为预测头颈鳞癌颈淋巴结转移的一个重要指标 ;VEGF可促进头颈鳞癌血管生成。     

Effects of the integrin-linked kinase inhibitor QLT0267 on squamous cell carcinoma of the head and neck

OBJECTIVE: To study the expression of integrin-linked kinase (ILK) in human squamous cell carcinoma of the head and neck (SCCHN) tumor specimens and cell lines and the efficacy of the novel small molecule QLT0267. DESIGN: Immunohistochemical analysis of 17 SCCHN tumor tissue specimens and 3 normal tongue tissue specimens for ILK expression and in vitro analysis of the effectiveness of QLT0267 on SCCHN cells. SETTING: Academic medical center. MAIN OUTCOME MEASURES: Expression levels of ILK in SCCHN tumor specimens and cell lines and the efficacy of QLT0267 in inhibiting cell growth and inducing apoptosis in SCCHN cell lines. RESULTS: Most SCCHN tumor specimens stained for ILK, whereas none of the 3 normal tongue tissue specimens stained for ILK. Integrin-linked kinase was expressed in all 6 SCCHN cell lines tested. In 4 pairs of normal and SCCHN tumor specimens, ILK expression and activity were higher in most tumor samples tested. A kinase assay showed that QLT0267 inhibited the ILK activity of 2 SCCHN cell lines (TU167 and MDA1986). Modified tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DNA fragmentation ladder, and TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end()labeling) assays showed that QLT0267 inhibited cell growth and induced apoptosis in these 2 cell lines. A dose-dependent decrease in Akt phosphorylation was observed for these 2 cell lines on treatment with QLT0267. CONCLUSIONS: Integrin-linked kinase is overexpressed in SCCHN tumor specimens. Targeting ILK with the small-molecule ILK inhibitor QLT0267 inhibits cell growth and induces apoptosis in SCCHN cell lines by reducing ILK activity and Akt phosphorylation. Integrin-linked kinase may be an attractive target for molecular therapy with which to enhance treatment of SCCHN.     

咽喉部鳞状细胞癌细胞系

咽喉部鳞状细胞癌是头颈外科常见恶性肿瘤,头颈部解剖结构复杂,原发部位不同的鳞状细胞癌发病率、诊疗方案不同,其发生的分子机制也可能存在差异。因此,选择适合的咽喉部鳞状细胞癌细胞系对于研究者选择适合的临床前研究模型,以及更好地理解肿瘤发生发展机制具有重要意义。总结了鼻咽、口咽、喉咽、喉四个解剖部位的鳞状细胞癌细胞系,以望对研究者选择适当的细胞系作为研究对象提供参考。     

Neck Dissection of Level IIb: Is it Really Necessary?

OBJECTIVES: To determine whether resection of level IIb is necessary in elective or therapeutic neck dissections. STUDY DESIGN: Prospective case series. METHODS: Level IIb nodes were analyzed for micrometastases as separate specimens in 160 neck dissections on 148 patients with squamous cell carcinoma of the head and neck. RESULTS: In 106 elective neck dissections (N0 necks) from upper aerodigestive tract (UADT) and skin/parotid squamous carcinoma primaries, level IIb was involved in 4.5% and 33%, respectively. In 54 therapeutic neck dissections (N+ necks) from UADT and skin/parotid squamous carcinoma primaries, level IIb was involved in 25% and 71%, respectively. Apart from skin/parotid squamous carcinoma primaries, level IIb was never involved unless level IIa was also involved. CONCLUSIONS: Level IIb nodes can be left in situ in UADT primary carcinomas in nontonsillar N0 necks without significantly compromising regional clearance of micrometastases.     

c-myc oncogene copy number in squamous carcinoma of the head and neck.

PURPOSE: Altered resident cellular genetic sequences (oncogenes) may result in malignant transformation, maintenance of tumor growth, and metastatic propensity. In this pilot study, we have elected to probe c-myc oncogene in evaluating specimens from human squamous cell carcinoma. MATERIALS AND METHODS: Samples were obtained from 24 patients with squamous cell carcinoma of the head and neck. The ratio of tumor DNA values to that of control DNA was used to estimate the c-myc copy number. RESULTS: Data from material obtained from eight patients was analyzed to the point of c-myc copy number. Tumors varied from stage II through IV. Five originated in the oral cavity and three in the larynx. Analysis of primary tumors demonstrated that two of eight had increased c-myc copy numbers. Histologically positive neck specimens were encountered in five of the study patients. Three demonstrated elevated c-myc copy numbers, two of which had had increased copy number at the primary site. CONCLUSION: This study confirms that c-myc amplification can be present in squamous cell carcinoma of the head and neck. c-myc Amplification may also be present in neck metastasis. Oncogene amplification in neck metastasis may indicate an increased metastatic propensity for individual tumor cells demonstrating c-myc amplification.