A comparative study of chemokine rantes detection using ELISA and Western blot.

Chemokines in biological sample are frequently found at very low level. Further, concentrations of chemokines which were measured in the same tissue detected with different methods often differ in literature. Therefore, RANTES concentrations were quantified by application of sandwich enzyme-linked immunosorbent assay (ELISA) and Western blot analyses. The sensitivity of ELISA was found to be much higher than that of Western blot. Additionally, the detection time differed considerably as well. For biological and biochemical characterization of chemokines it is essential to implement the optimal purification and detection techniques. With an understanding of the technical procedures and some pitfalls, chemokine detection can be applied more reliably.

Cyfra 21-1: a serological help for detection of distant metastases in head and neck cancer.

Local and neck recurrences of squamous cell carcinoma of the head and neck (SCCHN) can mostly be detected early when the patient has regular follow-ups. Distant metastases though usually remain undiscovered until they produce clinical symptoms. Since Cyfra 21-1 correlates with the tumor size and stage in SCCHN, we looked for possible connections between Cyfra 21-1 increases and the development of distant metastases. The sera of 830 patients with SCCHN were tested for Cyfra 21-1. The levels were compared with the clinical run of the patients. When Cyfra 21-1 levels rose above the threshold of 3.3 ng/ml (71 out of 830) staging procedures were performed. Tumor growth was found in 50 out of 71 patients with elevated Cyfra levels (70.4%). Cyfra serum levels in those cases either represented development of distant metastases (27 out of 71), or local and neck recurrences. The results of this study show that Cyfra 21-1 is a suitable and helpful serological parameter for the follow-up of patients with SCCHN. In the event of an elevation of Cyfra 21-1 above the threshold during follow-up, we would recommend the performance of a thoracal CT-scan and abdominal omi ultrasound as staging procedures.

mRNA differential display of human laryngeal carcinoma cells and corresponding benign keratinocytes: cloning versus PCR-amplification.

The mRNA expression profile of mucosal keratinocyte cell lines was compared to that of squamous cell carcinoma cell lines of the head and neck (UMSCC-10A and UTSCC-19A) by the differential display technique. Before the sequencing procedure, the differentially expressed fragments can be reamplified either by PCR amplification only or by PCR amplification combined with molecular cloning. In this study, these two methods are compared. Total RNA of both cell types was reverse transcribed into cDNA followed by amplification with PCR. After electrophoresis in a non-denaturing gel, differentially expressed fragments were isolated, reamplified, and sequenced. Reamplification was carried out following two different protocols: the first one included two additional rounds of PCR, the second one only one additional round of PCR followed by cloning. Differentially expressed fragments could be sequenced after reamplification with both methods. Different cloned recombinants of the same PCR pool showed sequence differences in one to three bases. On the other hand, amplification of the differentially expressed fragment by PCR alone was reproducible without any differences in sequence. But in the latter case, only one of the complementary strands could be sequenced. Differentially expressed mRNA fragments detected by differential display can be sequenced directly after reamplification in two additional rounds of PCR. This method is no adequate replacement for bacterial cloning, but it might be a suitable solution if cloning procedures cannot be performed.

Tracing human papillomavirus DNA in nasal polyps by polymerase chain reaction.

Human papillomavirus (HPV) infections are related to the genesis of various benign and malignant human neoplasias. The HPV types 16 and 18 seem to be causally related to the development of most squamous cell carcinoma of the anogenital tract and a proportion of carcinomas of the upper aerodigestive tract. The near 100% positivity of the HPV types 6 and 11 in laryngeal papillomatosis is well established. We investigated whether HPV also plays a role in non-neoplastic mucosal entities such as sinunasal polyposis, the genesis of which has been discussed as being triggered by viral infections. On DNA from 39 sinunasal polyps (33 patients), polymerase chain reaction (PCR) was performed using beta-globin primers for demonstration of amplifiable DNA in the tissue extracts. Consensus primers for the detection of several different HPV types were applied to the beta-globin-positive samples. The results were confirmed by Southern blot hybridization using consensus probes. Cycle sequencing was performed on the positive cases. All 39 samples showed positive signals for beta-globin. HPV-DNA investigations showed a slight positive signal in only 1 of the 39 investigated cases (2.6%). Further molecular investigations of this sample, including cycle sequencing, could not confirm this result. All the other tissue samples remained HPV-DNA-negative. Therefore, those HPV types readily detectable with the PCR primers and probes used are not frequently associated with sinunasal polyposis. The data confirm the hypothesis that HPV is correlated to a lesser extent to infectious mucosal lesions than to proliferative lesions. Furthermore, the results emphasize that the presence of HPV in specific lesions does not occur by chance, but represents a specific infection of the mucosa leading to proliferation and even to malignancy.

A new prognostic indicator for head and neck cancer--p53 serum antibodies?

BACKGROUND: p53 antibodies are a new serological parameter of unknown potential in patients with malignancies. Their occurrence has been described in various types of cancer patients and several studies in head and neck cancer patients and other cancer patient groups have indicated its prognostic value. MATERIAL AND METHODS: We investigated the incidence of p53 serum antibodies in 271 head and neck cancer patients with an ELISA and investigated a possible correlation with clinical parameters like tumor staging and grading. RESULTS: Sixty-seven head and neck cancer patients (24.7%) were seropositive for p53 antibodies. No correlation between p53 antibody status, tumor staging and grading was found. CONCLUSIONS: These results indicate that the occurrence of p53 antibodies does not correlate with the most relevant prognostic factors, rate of regional metastasis and primary tumor size, in patients with head and neck cancer.

Differentially expressed genes in head and neck cancer.

Carcinogenesis is considered a multistep process. To further elucidate involved genetic changes, the differential display method was applied to compare gene expression of head and neck carcinoma cells and normal keratinocytes from the upper aerodigestive tract. Total RNA was extracted from cultured squamous carcinoma cells and keratinocytes. mRNA was reverse transcribed into cDNA, amplified by PCR, and separated on a gel. Currently three DNA transcripts were identified with a length of 191 to 336 base pairs (bp) that were either expressed only by the keratinocytes or by the malignant cells. Differentially expressed DNA fragments of the carcinoma cells and the keratinocytes were cloned and sequenced. A gene bank database search identified one fragment expressed by the carcinoma cells as an unknown gene, another one found in the keratinocytes as probably a part of the human cell attachment domain, and the third one with homology to the mRNA of the human epidermal growth factor receptor (EGFR). Northern blot analysis confirmed the differential expression in the malignant cells or the keratinocytes. Differential display seems to confirm the well-known overexpression and up-regulation of the EGFR, the differential expression of the cell attachment domain may play a role as a cofactor in carcinogenesis of head and neck cancer, and the third unknown fragment is still under investigation to elucidate the role in carcinogenesis.

Prevalence of human papillomavirus in squamous cell carcinoma of the head and neck determined by polymerase chain reaction and Southern blot hybridization: proposal for optimized diagnostic requirements.

Human papillomaviruses (HPV) are discussed as cofactors in the carcinogenesis of squamous cell carcinoma of the head and neck (SCCHN). The prevalence of HPV infection in SCCHN is the subject of controversy since reported HPV prevalences range from less than 10% to almost 100%, depending mainly on the detection method employed. This study presents a realistic approximation to the real prevalence of HPV in SSCHN by applying polymerase chain reaction (PCR) and Southern blot hybridization (SBH), which are the most sensitive and specific HPV detection methods. Diagnostic procedures were optimized by applying a "hot-start" PCR protocol followed by a confirmatory SBH of the PCR products to reactions using both type-specific and consensus primers and probes. DNA of 75 tumour samples and 22 normal mucosa samples of the same patients were investigated. In 14 cases genomic SBH using complete HPV genomes as probes was performed additionally. HPV DNA was detected in 17/75 (22.7%) SCCHN specimens. HPV 16 was identified in four cases, HPV 45 in three cases, and HPV 6 and 18 in one case each. Hot-start PCR and SBH are the most reliable HPV detection methods, as they minimize both false-positive and false-negative results. With these methods, a HPV prevalence of 23% was achieved, which can be assumed to be representative for comparable study populations. The significant number of positives detected only by consensus primer PCR, along with the identification of HPV 45, indicate that further HPV types may play an important role in the genesis of SCCHN.

Optimized differential display and reamplification parameters for silver staining.

Differential display (DD) is a powerful instrument to detect differences in gene expression of malignant and normal cells. An optimized silver staining protocol was used to compare mRNA expression of keratinocytes and squamous cell carcinoma cells of the head and neck. Optimal concentration for upstream and downstream primer was 0.2 microM. Best primer concentrations for reamplification were between 40 and 60 pM. With this optimized protocol nearly 50 differentially expressed transcripts have been identified and differential display can be applied safer, easier, and faster, than by radioactive labeling.

Cut-off value determination of CYFRA 21-1 for squamous cell carcinomas of the head and neck (SCCHN).

BACKGROUND: CYFRA 21-1 was found to be a sensitive and specific tumor marker especially for squamous cell carcinomas of the lung. There is no reliable tumor marker for squamous cell carcinomas of the head and neck (SCCHN) available. METHODS: The amount of Cytokeratin (Ck) 19 in healthy tissue and tissues of benign and malignant tumors from pharynx, larynx and lung was compared utilizing a dot-blot assay. CYFRA 21-1 serum levels were determined prior to therapy in 132 healthy controls, in a reference group consisting of 158 patients with benign diseases of the head and neck and in 238 patients with a histologically proven SCCHN. RESULTS: Compared to lung tissue Ck 19 levels were similar only in specimen of SCCHN. Ck 19 content in normal and benign tissue from pharynx and larynx was lower than in normal lung tissue. CYFRA 21-1 serum levels were significantly higher in patients with SCCHN compared to the control or reference group. The cut-off value for CYFRA 21-1 determined at a 95%-specificity of the reference group was found to be 2.2 ng/ml. CONCLUSIONS: The Ck 19 level in healthy tissue of the upper aerodigestive tract was lower than in normal lung tissue. Corresponding to this finding the CYFRA 21-1 serum level in the reference group with benign diseases of the head and neck was lower than that of patients with benign lung diseases. The cut-off level of CYFRA 21-1 for patients with SCCHN was determined to be 2.2 ng/ml.

Head and neck cancer and p53-immunogenicity.

BACKGROUND: p53-mutations are of major importance in the development of human malignancies and occur frequently in head and neck cancer. The detection of serum p53-antibodies has been performed for a number of different cancers. For head and neck cancer though, the occurrence of serum p53-antibodies has not been determined so far. MATERIALS AND METHODS: A set of 82 sera from patients with squamous cell carcinomas of the head and neck were screened for circulating antibodies against p53 with ELISA. RESULTS: Of 82 patients 22% (n = 18) demonstrated p53-antibodies in their sera; the specificity for malignancy was 100%. CONCLUSIONS: As far as we know, this is the first study to reveal p53-antibodies in the sera of patients with SCCHN. The high incidence of positivity for p53-antibodies in this subset of cancer patients may give additional help in the diagnosis of this often disfiguring disease.

p53 serum antibodies as prognostic indicator in head and neck cancer.

p53 antibodies are a new serological parameter of unknown potential in patients with malignancies. Their occurrence has been described in various types of cancer patients. The mechanism underlying the immunization process is still unclear. We investigated the incidence of p53 serum antibodies in 143 head and neck cancer patients with an enzyme-linked immunosorbent assay. The posttherapy course of two matched study groups (n = 38 each), one p53-antibody-seropositive and one p53-antibody-seronegative, was followed up for 24 months. Thirty-nine head and neck cancer patients (27.3%) were seropositive for p53 antibodies. During the follow-up, the p53-antibody-seropositive patients accounted for more local tumor recurrences (n = 12 versus n = 8) and more tumor-related deaths (n = 11 versus n = 5) than did seronegative patients, and second primary tumors (n = 9 versus n = 0) occurred exclusively in seropositive patients. In total, therapy failures (recurrences, tumor-related deaths, second primaries) were observed in 17/38 cases (44.7%) in the p53-antibody-seropositive group and in 8/38 cases (21.1%) in the p53-antibody-seronegative group. These results, after a follow-up of 2 years, seem to indicate a prognostic value of p53 serum antibodies for therapy failure in patients with head and neck cancer.

High rate of p53 overexpression in head and neck carcinomas detected with a refined ELISA.

p53 mutations are amongst the most prevalent alterations in human cancer. Overexpression of p53 is usually caused by mutation and is observed in a high percentage of squamous cell carcinomas of the head and neck (SCCHN). Fifty two fresh samples of SCCHN were examined for p53 overexpression and 23 tumor-free tonsils served as controls. Using the monoclonal antibody Pab 1801 a refined ELISA technique was employed. In contrast to established ELISA procedures tumor tissue was pulverized at -80 degrees C prior to the actual ELISA (ELISA I). Comparative p53 detection was carried out by immunohistochemistry (IHC) and a commercially available ELISA kit (ELISA II). The modified ELISA I revealed p53 overexpression in 48 tumor samples (92%). p53 detection was obtained in 26 cases (50%) with ELISA II and with IHC 39 stained positive for p53 (75%). All of the controls were negative for p53 with ELISA and IHC. The p53 staining in IHC showed a significant correlation with the grading of the tumor. The ELISA results of the p53 overexpression did not show any correlation with tumor size or stage and rate of metastasis or other clinical parameters. This ELISA represents a more sensitive detection method of p53 than any other technique so far. It improves on former ELISA and IHC results on p53 overexpression in squamous cell carcinoma of the head and neck and underlines the involvement and the importance of the p53 tumor suppressor protein in carcinogenesis of head and neck cancer.