Analysis of p53 serum antibodies in patients with head and neck squamous cell carcinoma

BACKGROUND: Mutation of the p53 tumor suppressor gene (also known as TP53) often leads to the synthesis of p53 protein that has a longer than normal half-life. Mutant p53 protein that accumulates in tumor cell nuclei can be detected by means of immunohistochemical staining techniques. Serum antibodies directed against p53 protein (p53-Abs) have been detected in some cancer patients. PURPOSE: We assayed serum samples from 80 patients with head and neck squamous cell carcinoma (HNSCC) for the presence of p53-Abs, and we evaluated potential associations between the presence of these antibodies and other histopathologic and clinical features. METHODS: Serum was collected from each patient at the time of diagnosis. In addition, tumor biopsy specimens were obtained before the initiation of treatment. An enzyme-linked immunosorbent assay was used to detect p53-Abs. The accumulation of p53 protein in tumor cell nuclei was assessed immunohistochemically by use of the anti-p53 monoclonal antibody DO7. Patient treatment consisted of radiotherapy alone, primary chemotherapy followed by radiotherapy, or surgery and postoperative radiotherapy. Relapse-free and overall survival from the beginning of treatment were estimated by use of the Kaplan-Meier method; survival comparisons were made by use of the logrank statistic. Univariate and multivariate analyses were conducted to identify factors associated with survival. Reported P values are two-sided. RESULTS: Fifteen (18.8%) of the 80 patients had p53-Abs. Tumor cell nuclei in 43 (58.9%) of 73 assessable biopsy specimens exhibited strong p53 immunostaining. Patient treatment method and the accumulation of p53 protein in tumor cell nuclei were not associated with increased risks of relapse or death. In univariate analyses, advanced tumor stage (> T1 [TNM classification]) and the presence of p53-Abs were significantly associated with an increased risk of death (P for trend = .007 and P = .002, respectively), whereas advanced tumor stage, substantial regional lymph node involvement (> N1), and the presence of p53-Abs were associated with an increased risk of relapse (P for trend = .002, P = .02, and P < .0001, respectively). In multivariate analyses, advanced tumor stage and the presence of p53-Abs were significantly associated with increased risks of relapse (p for trend = .04 and P = .003, respectively) and death (P for trend = .04 and P = .03, respectively). At 2 years of follow-up, the overall survival proportion was 63% (95% confidence interval [CI] = 47%-80%) when no p53-Abs were detected compared with 29% (95% CI = 4%-54%) when p53-Abs were detected. Relapse-free survival at 2 years was 62% (95% CI = 49%-76%) if no p53-Abs were detected compared with 13% (95% CI = 0%-31%) if p53-Abs were detected. CONCLUSIONS AND IMPLICATIONS: The proportion of patients with HNSCC who have serum p53-Abs is smaller than that of patients exhibiting tumor cell accumulation of p53 protein. The presence of p53-Abs is significantly associated with increased risks of relapse and death.     

Expression of galectins in head and neck squamous cell carcinoma

BACKGROUND: Galectins are carbohydrate-binding proteins thought to be important for cell growth and differentiation, whose expression is altered in some tumors with aggressive phenotype. Our objective was to evaluate the expression of galectins in head and neck squamous cell carcinoma (HNSCC). METHODS: Fourteen HNSCC cell lines and four primary tumor specimens were evaluated using immunoblotting, and immunohistochemical analysis was performed on 35 primary HNSCCs. RESULTS: Galectin-1 and galectin-3 were expressed in most HNSCC cell lines and primary tumor specimens. Galectin-1 was detected in the basal layer of normal adjacent mucosa, in connective tissue stroma, and at the periphery of invasive tumor islands. Galectin-3 localized to superficial mucosal layers, and adjacent to keratin pearls in invasive carcinoma. CONCLUSIONS: Galectins are manifested in HNSCC tumors and are localized to the cell surface, where they may participate in cellular interactions. The expression pattern of galectins appears to be associated with degree of squamous differentiation, suggesting a potential role for galectins as biologic and differentiation markers in HNSCC.     

Serum levels of tumor necrosis factor in squamous cell carcinoma of the head and neck

Two experiments were conducted to study the effect(s) of Borrelia burgdorferi and its metabolites (toxicants?) on canine spermatozoa, using B burgdorferi type strain B-31 and ejaculates from 5 sexually mature dogs. In Experiment 1 the spirochetes were cocultured with semen and incubated under various conditions, and in Experiment 2 the spirochetes were sonicated to release the metabolites/toxicants. The sonicate was then cultured with the semen. The parameters investigated were kinematics and percentage of sperm motility, morphology, and sperm response to the hypoosmotic swelling test and acrosome reaction. There were no visible physical interaction between either dead or motile spirochetes and viable or dead spermatozoa. Neither the spirochetes per se nor their metabolites/toxicants had any significant adverse effect on the functional and morphological characteristics of the canine spermatozoa. It is possible that the exposure times for incubation were not long enough for metabolites or toxicants in the sonicate to significantly affect sperm characteristics. Some investigators have reported that B burgdorferi contain biologically active LPS-like endotoxins. It is also likely that storage denatures B burgdorferi metabolites and other intracellular products in the sonicate and, thus, negates any effects the medium or the sonicate might have on the spermatozoa. The apparent lack of effect suggests that either peripheral metabolism or action on other organ(s) may be required for deleterious action of the spirochetes and/or their toxicants on spermatozoa. It was concluded that B burgdorferi and/or metabolites/toxicants do not have any significant deleterious effects on the functional and morphological characteristics of the postspermatogenic spermatozoa. Interaction of the spirochetes with in vivo conditions may be needed to adversely affect spermatozoa.     

Aggressive cutaneous squamous cell carcinoma of the head and neck in patients with chronic lymphocytic leukaemia

We describe multiple cutaneous squamous cell carcinomas of the head and neck in five patients with chronic lymphocytic leukaemia (CLL). When associated with CLL, cutaneous squamous cell carcinomata behave in a much more aggressive manner than otherwise expected. Four patients developed local recurrence after primary treatment. All five patients developed lymph node metastases containing squamous cell carcinoma. Three of five patients (60 per cent) had multiple primary lesions. Whereas the increased incidence of second cancers in CLL and notably of skin cancers is documented, little has been written to describe the aggressive behaviour of these tumours. It is important, when treating these patients, to be aware of the high tendency towards local recurrence and lymph node metastasis and to consider an aggressive management plan and careful follow-up.     

Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma

PURPOSE: Standard therapies of head and neck squamous cell carcinoma (HNSCC) often cause profound morbidity and have not significantly improved survival over the last 30 years. Preclinical studies showed that adenoviral vector delivery of the wild-type p53 gene reduced tumor growth in mouse xenograft models. Our purpose was to ascertain the safety and therapeutic potential of adenoviral (Ad)-p53 in advanced HNSCC. PATIENTS AND METHODS: Patients with incurable recurrent local or regionally metastatic HNSCC received multiple intratumoral injections of Ad-p53, either with or without tumor resection. Patients were monitored for adverse events and antiadenoviral antibodies, tumors were monitored for response and p53 expression, and body fluids were analyzed for Ad-p53. RESULTS: Tumors of 33 patients were injected with doses of up to 1 x 10(11) plaque-forming units (pfu). No dose-limiting toxicity or serious adverse events were noted. p53 expression was detected in tumor biopsies despite antibody responses after Ad-p53 injections. Clinical efficacy could be evaluated in 17 patients with nonresectable tumors: two patients showed objective tumor regressions of greater than 50%, six patients showed stable disease for up to 3.5 months, and nine patients showed progressive disease. One resectable patient was considered a complete pathologic response. Ad-p53 was detected in blood and urine in a dose-dependent fashion, and in sputum. CONCLUSION: Patients were safely injected intratumorally with Ad-p53. Objective antitumor activity was detected in several patients. The infectious Ad-p53 in body fluids was asymptomatic, and suggests that systemic or regional treatment may be tolerable. These results suggest the further investigation of Ad-p53 as a therapeutic agent for patients with HNSCC.     

Microsatellite instability in squamous cell carcinoma of the head and neck

Genomic instability or microsatellite instability (MI) in simple repeated sequences was initially recognised in colonic carcinomas and subsequently in other tumours. MI has been associated with mutations in genes concerned with replication and DNA repair. We investigated 34 microsatellite markers in squamous cell carcinoma of the head and neck (SCCHN). Fifty-six tumours, were studied, of which 25 were investigated with ten or more microsatellite markers. In this study we consider two or more microsatellite alterations in a tumour to be diagnostic of MI. We demonstrated that 7/25 (28%) of the tumours had MI at two or more loci and three of these tumours exhibited evidence of 20 or more loci with MI. No correlations were found between MI and previous treatment, site, histological differentiation, positive nodes at pathology, a history of alcohol intake or survival. MI has been demonstrated in T1N0 stage tumours, indicating that these changes may occur early in the disease process. A negative correlation was found between MI and a history of smoking (P = 0.02). Two or more markers of MI were found in three of four non-smokers compared with one of 13 in the smoking group of patients, which suggests a novel mechanism of carcinogenesis in non-smokers.     

Radiologic diagnosis and staging of head and neck squamous cell carcinoma

The predominant extracranial head and neck cancer in adults is squamous cell carcinoma. The purpose of this article is to discuss the radiographic evaluation of these patients with computed tomography (CT) or magnetic resonance (MR) imaging prior to therapeutic intervention. Specific focus is given to the efficacy of CT and MR imaging, as an adjunct to clinical staging, for evaluation of the primary tumor, and metastatic adenopathy. MR imaging, because of its improved soft tissue contrast and multiplanar capability, is probably superior to CT for evaluation of the primary tumor in patients with squamous cell carcinoma. CT, however, remains the gold standard for identifying metastatic adenopathy and in most institutions remains the study of choice for evaluating this patient population.     

Vascular endothelial growth factor expression in head and neck squamous cell carcinoma

BACKGROUND: Angiogenesis is an essential process required for growth and metastasis in cancer. In breast, gastric, and prostate cancer, vascular endothelial growth factor (VEGF) has been implicated in angiogenesis; however, little is known about VEGF in HNSCC. In this study, we hypothesize that VEGF is present in elevated levels in HNSCC and may therefore play a role in promoting angiogenesis. METHODS: We obtained tumor tissue from 63 HNSCC patients undergoing primary resection. All tissue samples were analyzed by immunohistochemistry (IHC) techniques for the presence and localization of VEGF; however, only 36 had sufficient amounts of tissue for quantitative analysis of VEGF by ELISA. Nine control specimens taken from patients undergoing uvulopalatopharyngoplasty were also analyzed. RESULTS: In all 63 of our patient samples we found VEGF to be present and localized to the cancer cells and endothelial cells. The poorly differentiated cancer cells stained more intensely in comparison with the well-differentiated ones. There was a 20-fold increase in the patient levels when compared with controls levels (P > or =0.05). Analysis by enzyme-linked immunosorbent assay revealed elevated mean levels of VEGF (241 +/- 326 pg/mg total protein [TP]) with a range of 2 to 1484 pg/mg TP. The control specimens had mean levels of 13 +/- 11 pg/mg TP and a range of 1 to 78 pg/mg TP. Patients who exhibited higher levels of VEGF tended to have a higher rate of disease recurrence (P < or =0.048) and shorter disease-free interval (P < or =0.05). CONCLUSIONS: The expression of VEGF in elevated levels in the HNSCC tumor microenvironment appears to be associated with more aggressive disease. Based on our results, VEGF may be an important angiogenic factor associated with cancer cells and endothelial cells in HNSCC. Further studies are needed to better define the role of VEGF in HNSCC and its role as a potential target for therapeutic intervention.     

Normalization of EGFR mRNA levels following restoration of wild-type p53 in a head and neck squamous cell carcinoma cell line

Hapalotrema mehrai Rao, 1976 and Hapalotrema postorchis Rao, 1976 (Digenea: Spirorchidae) are redescribed from the heart and pulmonary arteries of the green turtle, Chelonia mydas, from Moreton Bay in south-eastern Queensland. Hapalotrema pambanensis Gupta and Mehrotra, 1981 from C. mydas in India is made a synonym of H. mehrai. Hapalotrema dorsopora Dailey, Fast and Balazs, 1993 from C. mydas from Hawaii was described with a dorsally opening uterine pore, but this is found to be the opening of Laurer's canal; therefore H. dorsopora is also made a synonym of H. mehrai. In addition to differences in the numbers of testes and general dimensions, H. mehrai and H. postorchis differ in the development of Laurer's canal and in the absence of a canalicular seminal receptacle in H. postorchis.     

Phase II trial of paclitaxel, ifosfamide, and cisplatin in patients with recurrent head and neck squamous cell carcinoma

PURPOSE: To assess the activity and toxicity profile of combined taxol (paclitaxel), ifosfamide, and platinum (cisplatin) (TIP) in patients with recurrent or metastatic squamous cell carcinoma (SCC) of the head and neck. PATIENTS AND METHODS: Recurrent or metastatic head and neck SCC patients received paclitaxel 175 mg/m2 in a 3-hour infusion on day 1; ifosfamide 1,000 mg/m2 in a 2-hour infusion on days 1 through 3; mesna 600 mg/m2 on days 1 through 3; and cisplatin 60 mg/m2 on day 1, repeated every 3 to 4 weeks. All were premedicated with dexamethasone, diphenhydramine, and cimetidine. Prophylactic hematopoietic growth factors were not permitted. RESULTS: Fifty-two patients were assessable for response and toxicity; 53 for survival (local-regional recurrence alone in 57% and distant metastasis with or without local-regional recurrence in 43%). Overall response rate was 58% (30 of 52) of patients; complete response rate was 17% (nine of 52) of patients, with six complete responses that continued for a median 15.7+ months. Median follow-up of all patients was 17.7 months. Median survival was 8.8 months (95% confidence interval [CI] 8.1 to 17.5 months). Toxicity was relatively well tolerated and caused no deaths. The most frequent moderate-to-severe toxicity (90% of patients) was transient grades 3 to 4 neutropenia; neutropenic fever occurred in 27%. Grade 3 peripheral neuropathy occurred in three patients, none had grade 4. Grade 3 mucositis occurred in only one patient, none had grade 4. CONCLUSION: TIP had major activity in this setting, with a 58% objective response rate, 17% complete response rate, durable complete responses (six of nine persisting), and relatively well-tolerated toxicity, with no toxic deaths. The activity of TIP, a novel taxol-cisplatin-based regimen, in recurrent or metastatic head and neck SCC should be confirmed in a phase III trial.     

MR imaging in squamous cell carcinoma of the head and neck with no palpable lymph nodes

PURPOSE: To assess the efficacy of MR imaging in the detection of lymph node metastasis in patients with no palpable lymph nodes ("N0 neck") who have squamous cell carcinoma of the head and neck region. MATERIAL AND METHODS: MR neck imagings in 18 patients who underwent neck dissection (bilaterally in 2) for squamous cell carcinoma of the head and neck region were examined preoperatively for the purpose of detecting lymph node metastases. The imaging features taken into consideration were: size (cutoff point 10 mm), grouping, presence of central necrosis, and appearance of extracapsular spread. The MR examinations comprised spin-echo T1- and T2-weighted sequences. The MR findings were compared with those of surgery and histopathological examination. RESULTS: MR suggested metastatic lymph node involvement in 5 necks. In 2 of these, central necrosis was seen in the enlarged lymph nodes. In a third, a grouping of the lymph nodes was noted. Extracapsular spread was not present. Histopathological examination revealed metastatic lymph nodes in 7 of the 20 necks, the rate of clinically occult disease being 35%, and 4 of them had been accurately graded by MR. There was one false-positive MR examination. The MR sensitivity was 57.1% and specificity 92.3%. CONCLUSION: MR may reveal metastatic lymph nodes in patients with no clinical evidence of metastasis. However, conventional MR techniques are not always sufficient for decision-making on surgery in cases of "N0 neck".     

Prognostic factors of cervical lymph node metastasis in head and neck squamous cell carcinoma

AIMS AND BACKGROUND: The metastatic spread of squamous cell carcinoma of the head and neck (SCCHN) to the cervical lymph nodes is a negative prognostic factor in terms of survival. We have used multivariate analysis to identify the possible prognostic significance of a number of clinical and pathological characteristics in relation to possible involvement of the cervical lymph nodes in a series of 396 patients. METHOD: 396 patients with SCCHN were studied. Variables regarding the patient, the carcinoma and histology were analysed by multivariate analysis using BMDP's PLR programme. RESULTS: Some variables appear to represent predisposing factors for tumor spread to the lymph nodes: tumor site (supraglottic larynx: P = 0.005; base of the tongue: P = 0.02; hypopharynx: P = 0.02), grading (P = 0.001), and a number of histological parameters (lower degree of histological differentiation: P = 0.001; vascular permeation: P = 0.04; perineural invasion: P < 0.05; prevalently plasmocytic infiltrate: P < 0.05). CONCLUSION: The identification of cases at risk for metastasis can be improved by the assessment of prognostic factors, with a consequent improvement in treatment strategies.     

Nitric oxide synthase activity in human squamous cell carcinoma of the head and neck

A continuous fluorometric assay for tryptophan hydroxylase activity based on the different spectral characteristics of tryptophan and 5-hydroxytryptophan is presented. Hydroxylation of tryptophan at the 5-position results in a large increase in the fluorescence of the molecule. The assay selectively monitors the fluorescence yield of 5-hydroxytryptophan by exciting the reaction mix at 300 nm. The rate of increase of the emission signal was found to be directly proportional to the enzyme concentration. Inner filter effects due to quinonoid dihydropterin accumulation were eliminated by the inclusion of a thiol reductant. Activity measured using this assay method was found to be the same as that determined by established discontinuous HPLC assay methods. The application of the assay to routine activity measurements and to steady-state determinations with the substrates tryptophan and tetrahydropterin is described.     

Predictive and prognostic markers in a series of patients with head and neck squamous cell invasive carcinoma treated with concurrent chemoradiation therapy

It has been proposed that diverse anticancer drugs and radiation therapy may induce a mode of cell death with the characteristics of apoptosis. Since apoptosis is under the control of several oncogenes, we analyzed the expression of the protein encoded by the proto-oncogenes bcl-2 and p53. Furthermore, we studied cell proliferation [using PC-10 mAb to proliferating cell nuclear antigen (PCNA)] and vascularization [using the CD-31 mAb and by counting intratumoral microvessel density (IMD)] using immunocytochemistry. A series of 73 patients with clinical stage II-IV squamous cell invasive carcinoma of the head and neck (H&N) were treated with concurrent chemoradiation therapy (cisplatin, 80 mg/m2, versus carboplatin, 375 mg/m2, three times every 3 weeks and a total dose of radiation therapy of 64 Gy in 6-8 weeks). We correlated the expression of these markers, determined prior to treatment, with response to the therapy and prognosis. Bcl-2 protein was expressed in 37.4% of the carcinomas (25/67 evaluable), and it was not significantly associated with any other feature studied. Forty (56. 4%) of the 71 carcinomas evaluable for p53 were p53 positive; the median IMD was 38 microvessels/field at the hot spot (range, 18-80), and the median percentage of nuclei labeled by the PC-10 mAb was 50% (range, 0-95%). In the univariate analysis, regional lymph node negativity (P = 0.016), good performance status (PS) (PS >/= 90; P = 0.044), bcl-2 positivity (P = 0.070), and low vascularization (P = 0. 085) were significantly associated with a higher probability of complete remission. In the multivariate analysis (final model), only IMD (continuous variable; P = 0.045) and PS (P = 0.017) retained significance. As far as prognosis is concerned, in the univariate analysis, patients with tumors with low histological grading (grades 1-2; P = 0.006), p53 negative (P = 0.09), bcl-2 positive (P = 0.08), and high PCNA labeling (P = 0.06) had a significantly better disease-free survival. In the multivariate analysis, only grading (P = 0.003) and p53 (P = 0.04) retained significance for disease-free survival. For overall survival, in the univariate analysis, the following markers were significantly prognostic when only deaths due to progression are considered: response to therapy (P = 0.00001), PS (P = 0.04), nodal status (P = 0.028), PCNA (P = 0.04), p53 (P = 0. 08), and grading (P = 0.01). In the multivariate analysis, only patients who achieved complete response (P = 0.00002), high PCNA values (P = 0.002), and low histological grading (P = 0.01) retained a statistically significant probability of better overall survival. Our results suggest that in this series of H&N cancer patients the markers capable of predicting response to therapy are distinct from those associated with prognosis, once the remission has been achieved. This information is potentially useful to the clinician for developing a more rational therapeutic approach for H&N cancer patients eligible for concurrent chemoradiation therapy.     

Effects of dietary anticarcinogens on rat gastrointestinal glutathione S-transferase theta 1-1 levels

Several naturally occurring food components or non-steroidal anti-inflammatory drugs (NSAIDs) may reduce gastrointestinal cancer rates. Recently we have shown that dietary administration of such compounds enhanced the glutathione S-transferase (GST) enzyme activity and class alpha, mu and pi isoenzyme levels in the rat gastrointestinal tract. Elevation of the levels of GSTs, a family of biotransformation enzymes with many functions such as detoxification of carcinogens, might be one of the mechanisms that lead to cancer prevention. We therefore investigated whether the anticarcinogens alpha-angelicalactone, alpha-tocopherol, beta-carotene, coumarin, ellagic acid, flavone, indole-3-carbinol, d-limonene, oltipraz, phenethylisothiocyanate (PEITC) and the sulphoraphane analogue compound-30 affect gastrointestinal rGSTT1-1 protein levels in male Wistar rats. rGSTT1-1 protein levels were determined in cytosolic fractions of liver and oesophageal-, gastric-, small intestinal- and colonic mucosa by densitometrical analyses of western blots after immunodetection with an anti human GSTT1-1 monoclonal antibody, that cross-reacts with rGSTT1-1. In control Wistar rats, gastrointestinal rGSTT1-1 protein levels were highest in the liver and decreased in the order liver > stomach > colon > oesophagus > small intestine. Gastric rGSTT1-1 protein levels were enhanced by alpha-angelicalactone, alpha-tocopherol, coumarin, ellagic acid, oltipraz, PEITC and the sulphoraphane analogue compound-30. Oesophageal rGSTT1-1 protein levels were elevated by a-angelicalactone and coumarin, whereas colonic rGSTT1-1 protein levels were elevated by coumarin. Ellagic acid, on the other hand, reduced hepatic rGSTT1-1 protein levels to 53% of the control. In conclusion, dietary anticarcinogens are capable of inducing rGSTT1-1 protein levels in the rat gastrointestinal tract, and are most pronounced in the stomach. Enhanced rGSTT1-1 protein levels might lead to an increase of enzyme activity and to a more efficient detoxification of carcinogens and thus could contribute to prevention of carcinogenesis.