p53 gene mutations and HPV infection in primary head and neck squamous cell carcinomas do not correlate with overall survival: a long-term follow-up study

We analyzed specimens of head and neck squamous cell carcinomas (HNSCC) from 110 patients for p53 gene mutations, and 92 of them for human papillomavirus (HPV) infection, in order to evaluate the prognostic significance of these factors by comparison with clinical follow-up data. Mutations within the exons 5 to 8 of the p53 gene were found in 48 tumors (44%). Sequencing revealed in most cases mis-sense mutations (16/21). Frequency of p53 gene mutations was not related to the tumor stage or the presence of lymph node metastases. Of the 46 tumors that were analyzed by immunohistochemistry, 26 stained positively (56%). The number of positively stained nuclei increased slightly with decreasing differentiation of the tumors, whereas no correlation was found between tumor stage and immunoreactivity. An infection with the high-risk HPV types 16 and 18 could be detected in 39/92 tumor specimens (42%). Follow-up data were obtained from 99 patients within a range of 2 to 112 months. No dependence of overall survival on the presence of p53 gene mutations or HPV infection could be observed. The absence of statistically significant correlations between p53 gene mutation and progressive disease, however, does not deny its putative relevance in early phases of tumor development.     

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.     

Overexpression of the p53 gene in primary and metastatic head and neck carcinomas

The p53 gene has been correlated with disease progression in a number of human malignancies, and p53 abnormalities are found in a high percentage of head and neck squamous cell carcinomas. The objectives of this study were 1. to correlate p53 expression with disease progression in squamous cell carcinoma of the head and neck (SCCHN), and 2. to determine whether there are site-specific differences in p53 expression. Primary lesions and/or lymph node metastases from 147 patients with invasive SCCHN were immunostained for p53 overexpression. Expression of p53 was similar (42% versus 43%) in primary lesions and lymph node metastases. Expression also did not vary significantly by site in the head and neck. In conclusion, increased p53 expression did not correlate with disease progression in our series of patients with invasive SCCHN. The finding of a lack of increased expression with disease spread to lymph nodes supports the belief that p53 alterations occur early in head and neck carcinogenesis.     

Functional characterization in vivo of mutant p53 molecules derived from squamous cell carcinomas of the head and neck

Loss of wild-type p53, either through deletion or mutation, has been demonstrated in most squamous cell carcinomas of the head and neck (HNSCC). Whether these mutant molecules contribute to tumor progression purely through loss of wild-type functions or by growth-promoting mechanisms, however, remains unclear. To begin to address these issues, we isolated a series of p53 cDNAs from HNSCC cell lines that contain missense or nonsense point mutations, insertions, or deletions. The ability of each of these molecules to transform NIH/3T3 cells to a malignant phenotype was assessed by stable transfection and expression under the control of a strong heterologous promoter. NIH/3T3 cells transfected with pLTR6p53, which harbors an H179L missense mutation, formed large tumors rapidly (in less than 4 wk) when transplanted to athymic mice, as did cells expressing pLTR13p53, which had undergone a V173F missense mutation and an in-frame deletion of 48 bp between codons 208 and 223. Cells transfected with pLTR17p53, predicted from the nucleotide sequence to encode a severely truncated p53 corresponding to the N-terminal 56 amino acids, also formed tumors. Cells transfected with pLTR15p53, which was predicted to encode a less severely truncated molecule, formed much smaller tumors and at lower frequencies. NIH/3T3 cells transfected with pLTR12p53 (exon 7 splice donor mutant), pLTRwtp53 (wild-type p53), or vector alone failed to form tumors for up to 2 mo after transplantation. pLTR6p53-transfected cells exhibited a highly malignant phenotype with invasion of regional lymph nodes, mediastinal and lung metastases, invasion of the abdominal wall, and dissemination throughout the peritoneal cavity. Histological assessment of the tumors revealed intensely vascularized fibrosarcomas with numerous cellular atypia, including frequent and aberrant mitoses. Tumor explants were recultured, and northern blot analysis of cellular RNA confirmed that the expression of exogenous p53 was maintained in each case. These data indicate that different p53 mutants contribute to tumorigenesis by specific mechanisms. Furthermore, the results obtained by using the pLTR17p53 transfectants imply that some truncated molecules may overcome the effects of wild-type p53 to contribute to malignancy.     

Genotypic analysis of tumor suppressor genes PTEN/MMAC1 and p53 in head and neck squamous cell carcinomas

OBJECTIVES: Tumor suppressor gene mutations in both p53 and PTEN/MMAC1 genomic DNA have been detected in many types of cancer. The purpose of this study was to investigate the presence and importance of PTEN/MMAC1 mutations in squamous cell carcinomas. METHODS: Exons of each gene were amplified after polymerase chain reaction (PCR) using genomic DNA derived from cell lines of squamous cell carcinoma of the head and neck (SCCHN) and snap-frozen biopsy specimens from primary established head and neck tumors. The amplified and purified DNA was then sequenced directly. RESULT: As anticipated, point mutations of the p53 gene were found in 80% of cell lines examined. A single base mutation in codon 151 was found in six of 10 cell lines studied. PTEN/MMAC1 gene mutations were found in neither the cell lines tested nor the tumor biopsy samples. CONCLUSION: This study, as well as a large volume of data, confirms that mutations of the p53 gene are frequent events in head and neck cancer cell lines. Although PTEN/MMAC1 gene mutations have been found in a variety of carcinomas, this gene was not found to be mutated in SCCHN cell lines or in primary squamous cell carcinomas of the head and neck. This information is useful for further studies of mutations in these cell lines.     

Six primary squamous cell carcinomas of the head and neck

Estrogens have a beneficial effect on atherosclerosis and osteoporosis after menopause, but their exact mechanism of action is still unknown. The aim of the present study was to investigate the effects of estradiol and its metabolites catechol estrogens on arachidonic acid metabolism in vitro. Estradiol had no effect on arachidonic acid metabolism up to 33 microM in A23187-stimulated human whole blood. All catechol estrogens (2-hydroxyestradiol, 2-hydroxyestrone, 4-hydroxyestradiol and 4-hydroxyestrone) had similar kinds of actions on arachidonic acid metabolism, being over ten times more potent inhibitors of leukotriene synthesis (IC50 values 0.044-0.16 microM) than thromboxane (IC50 values 0.99-2.1 microM) and prostaglandin E2 synthesis (IC50 values 0.84-5.5 microM). It is suggested that some of the protective actions of estrogens--e.g., on atherosclerosis and osteoporosis--may be related to the inhibition of leukotriene synthesis by catechol estrogens.     

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.     

Gene mutations and increased levels of p53 protein in human squamous cell carcinomas and their cell lines

Using immunocytochemical and Western blotting techniques we have demonstrated the presence of abnormally high levels of p53 protein in 8/24 (33%) of human squamous cell carcinomas (SCC) and 9/18 (50%) of SCC cell lines. There was a correlation between the immunocytochemical results obtained with eight SCC samples and their corresponding cell lines. Direct sequencing of PCR-amplified, reverse transcribed, p53 mRNA confirmed the expression of point mutations in six of the positive cell lines and detected in-frame deletions in two others. We also detected two stop mutations and three out-of-frame deletions in five lines which did not express elevated levels of p53 protein. Several of the mutations found in SCC of the tongue (3/7) were in a region (codons 144-166) previously identified as being a p53 mutational hot spot in non-small cell lung tumours (Mitsudomi et al., 1992). In 11/13 cases only the mutant alleles were expressed suggesting loss or reduced expression of the wild type alleles in these cases. Six of the mutations were also detected in the SCCs from which the lines were derived, strongly suggesting that the mutations occurred, and were selected, in vivo. The 12th mutation GTG-->GGG (valine-->glycine) at codon 216 was expressed in line SCC-12 clone B along with an apparently normal p53 allele and is to our knowledge a novel mutation. Line BICR-19 also expressed a normal p53 allele in addition to one where exon 10 was deleted. Additionally 15 of the SCC lines (including all of those which did not show elevated p53 protein levels) were screened for the presence of human papillomavirus types 16 and 18 and were found to be negative. These results are discussed in relation to the pathogenesis of SCC and the immortalisation of human keratinocytes in vitro.     

Involvement of p53 gene mutations in human endometrial carcinomas

Mutations in the p53 gene are associated with a wide variety of human malignancies. Point mutation in one allele and loss of the remaining one generally lead to inactivation of p53 protein. A high frequency of allelic losses corresponding to the 17p13.3 region that contained the p53 gene sequence was also noted in human endometrial carcinoma. Thus, in order to confirm involvement of the p53 gene in endometrial carcinogenesis, we searched for nucleotide sequence change in this gene in 42 endometrial carcinomas that had been subjected to previous LOH analyses. Using the polymerase-chain-reaction-single-strand conformation polymorphism (PCR-SSCP) method, we detected p53 gene mutations in 4 specimens. Two adenocarcinomas with allelic loss on 17p contained a mutant p53 gene in the allele that was retained. One specimen with a p53 gene mutation contained a 17q deletion but was uninformative for LOH on 17p. p53 gene mutation was also noted in the remaining stage-I carcinoma, though the 17p deletion was not detected in the previous LOH examination. However, 5 specimens with the LOH on 17p retained the wild-type p53 gene. In the remaining 33 specimens, both alleles of p53 gene seemed to be normal. The mutations observed in 2 specimens (cases 10 and 24), involving C-to-T and T-to-G substitutions, were located in a highly conserved region. However, the mutations identified in the remaining 2 cases (29 and 35) were at regions positioned outside conserved stretches.     

Prognostic significance of p53 gene mutations in squamous cell carcinoma of the lung

The aim of the present study was to analyze the prevalence and clinical importance of p53 gene mutations in surgically treated squamous cell lung carcinoma. Sixty patients were included. Fifty-one patients in stages I to IIIa were submitted to radical resection. Twenty-five samples tested positive for the p53 immunohistochemistry assay, and were analyzed for p53 gene mutations. Eleven mutations were found. Patients harboring p53 gene mutations suffered a higher incidence of recurrence and a higher mortality rate. Disease-free interval and overall survival were shorter for patients with mutated p53 gene (p=0.03 and p=0.005, respectively).     

An unexpected spectrum of p53 mutations from squamous cell carcinomas in psoriasis patients treated with PUVA

Photochemotherapy employing 8-methoxypsoralen and long-wavelength ultraviolet radiation (UVA, 320-400 nm) is widely used in the treatment of psoriasis. The photoactivation of psoralens in skin cells leads to formation of DNA photoadducts which may be responsible, at least in part, for the efficacy of these photochemotherapies. However, mutations arising from these adducts may also lead to the well-characterized increased incidence of squamous cell carcinoma. Mutations in the p53 tumor suppressor gene have been detected in many human cancers. To determine whether p53 mutations occur in squamous cell carcinomas in PUVA patients, PCR was used to amplify the exons (5-9) in which other studies have found a high frequency of point mutations. Gel electrophoresis was used to detect single-strand conformational polymorphisms. Aberrantly migrating bands were excised, reamplified and sequenced. Thirty-four specimens from 10 patients were examined. Specimens from one patient who had received no phototherapy as well as from normal controls were also analyzed. Five of the 10 patients showed at least one p53 mutation. In contrast to previously reported psoralen-induced p53 mutations in mice, the expected psoralen type mutations at alternating AT sites were not detected. All but two of the altered sequences occurred at dipyrimidine sites which is typical of solar type mutations. Two C-->T mutations and two dipyrimidine mutations (CC-->TT) were found. Other mutations included: C-->G, G-->T, C-->A and an 18 bp deletion. A review of therapeutic history of these patients showed that some had also received UVB phototherapy. Furthermore, because sunlight is thought to be beneficial for psoriasis, nontherapeutic, casual UVB exposure cannot be excluded. Our observations suggest that the SCC may have arisen from the solar mutations and that PUVA may enhance tumor progression or immune suppression.     

Complex karyotypes in flow cytometrically DNA-diploid squamous cell carcinomas of the head and neck

In squamous cell carcinoma of the head and neck (SCCHN), DNA ploidy as determined by flow cytometry (FCM) has been found to yield prognostic information but only for tumours at oral sites. Cytogenetic findings have indicated complex karyotype to be a correlate of poor clinical outcome. In the present study, 73 SCCHN were investigated with the two techniques. Aneuploid cell populations were identified in 49 (67%) cases by FCM but in only 21 (29%) cases by cytogenetic analysis. The chromosome index (CI), calculated as the mean chromosome number divided by 46, was compared with the respective DNA index (DI) obtained by FCM in 15 tumours, non-diploid according to both techniques, DI being systematically 12% higher than CI in this subgroup. Eight (33%) of the 24 tumours diploid according to FCM had complex karyotypes, three of the tumours being cytogenetically hypodiploid, three diploid and two non-diploid. The findings in the present study may partly explain the low prognostic value of ploidy status as assessed by FCM that has been observed in SCCHN. In addition, we conclude that FCM yields information of the genetic changes that is too unspecific, and that cytogenetic analysis shows a high rate of unsuccessful investigations, thus diminishing the value of the two methods as prognostic factors in SCCHN.     

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.     

Distinct patterns of chromosomal alterations in high- and low-grade head and neck squamous cell carcinomas

Comparative genomic hybridization was performed on 30 primary head and neck squamous cell carcinomas. Fractional or entire DNA loss of chromosome 3p was a basic finding that occurred in 29 cases (97%). Additional DNA underrepresentations were observed in more than 50% of the cases on chromosomes 1p, 4, 5q, 6q, 8p, 9p, 11q, 13q, 18q, and 21q. Deletions on chromosomes 3p, 13q, and 17p were confirmed by loss of heterozygosity analysis. Entire or partial DNA copy number increases were identified for chromosome 3q in 26 cases (87%) with high-level amplifications at 3q24 and 3q27-qter. Overrepresentations were found in decreasing order of frequency at 11q13 (70%), 8q (57%), 19q (50%), 19p (47%), and 17q (47%). The use of comparative genomic hybridization superkaryograms of the group of well-differentiated carcinomas (G1) indicated that the deletions on chromosomes 3p and 9p along with the overrepresentation of 3q are associated with early tumor development. Accordingly, the undifferentiated tumors (G3) were characterized by additional deletions on chromosomes 4q, 8p, 11q, 13q, 18q, and 21q and overrepresentations on 1pter, 11q13, 19, and 22q, suggesting that these changes are preferentially associated with tumor progression.     

Heterogeneity of squamous cell carcinomas of the head and neck in relation to clinicopathological variables

We analysed 32 primary, 8 recurrent and 16 metastatic squamous cell carcinomas of the head and neck by flow cytometry to assess tumoural heterogeneity. Intratumoural heterogeneity was found in 9 of 30 primary tumours (30%). All heterogeneous tumours had diploid and aneuploid cell lines. DNA indices were different from those of the primary tumours in 3 of the 8 recurrent lesions (38%) and in 8 of the 12 metastatic lesions (67%). The results indicate that new cell lines evolve during the process of recurrence and metastasis. The incidence of recurrence, metastasis, and heterogeneity increased with the size of the tumour. Histologically, there were tumours in which the degree of differentiation and mode of invasion of primary lesions were not consistent with the results of flow cytometry in both homogeneous and heterogeneous tumours. In conclusion, multiple sampling for flow cytometric analysis is essential for better characterization of oral carcinomas.     

Different KGF expression in squamous cell carcinomas of the head and neck and in normal mucosa

Inducible nitric oxide synthase (iNOS) and TGF-beta were localized by immunocytochemistry in skin lesions from patients across the leprosy spectrum, and from patients undergoing reversal reaction. iNOS expression was highest at the tuberculoid pole of the spectrum, and increased during reversal reaction. TGF-beta was observed throughout the leprosy spectrum, but was highest at the lepromatous pole. Levels of TGF-beta decreased during reversal reaction. Reduced levels of TGF-beta may contribute to unregulated inflammatory responses during reactional episodes.     

Late p53 mutation in head and neck oncogenesis

p53 mutation are currently recognized as the commonest genetic event in human tumors. In this paper are studied, through immunochemistry, the p53 protein expression of 25 carriers from squamous cell carcinoma of the larynx and hypopharynx as well in the non tumoral mucosa of the larynx removed and corresponding metastases. Because of the great overexpression of this protein among tumors and its metastases, aside with the low expression in normal far-off mucous membranes of the studied growths, the AA. draw out the conclusion that p53 mutation is a late event by neck and head oncogenesis.     

p21WAF1/Cip1 expression is associated with cell differentiation but not with p53 mutations in squamous cell carcinomas of the larynx

The anti-metastatic effect of Z-100, an immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis, was investigated in mice bearing B16 melanoma cells. Treatment of BF10 mice implanted with high metastatic B16F10 melanoma cells with a 10 mg/kg dose of Z-100 resulted in the reduction of experimental pulmonary metastasis as compared with that of BF10 mice treated with saline. The number of pulmonary metastatic colonies in BF1 mice (mice implanted with low metastatic B16F1 melanoma cells) was greatly increased after the inoculation of CD4+ CD11b+ CD281+ TCR alphabeta+ type 2 T cells (F10-Th2 cells) derived from BF10 mice, while only a few metastatic colonies were demonstrated in lungs of BF1 mice inoculated with naive CD4+ T cells. However, the numbers of metastatic colonies in BF1 mice were not increased when they were inoculated with the F10-Th2 cell fraction derived from Z-100-treated BF10 mice and the generation of F10-Th2 cells in BF10 mice was effectively suppressed by the Z-100 treatment. These results suggest that Z-100 inhibits pulmonary metastasis of B16 melanoma through the regulation of tumor-associated Th2 cells, which are a key cell in the acceleration of tumor metastasis.