MMP and TIMP gene expression in head and neck squamous cell carcinomas and adjacent tissues.

OBJECTIVE: To compare the frequency of gene expression of matrix metalloproteinases (MMP) stromelysins -1, -2 and -3 (MMP-3, -10, and -11), matrilysin (MMP-7), MTI-MMP (MMP-14), and of TIMPs (Tissue Inhibitors of MMPs) -1, -2, -3 and -4 in head and neck squamous cell carcinomas with those of matched adjacent normal tissues. MATERIALS AND METHODS: The present study included 20 surgically removed head and neck squamous cell carcinomas, seven of which were accompanied by matched adjacent oral mucosa excised from the border of the specimens outside the tumor area. RNA isolated from tumors and control samples was subjected to RT-PCR using primers specific for MMP-3, -7, -10, -11 and -14 and for TIMPs -1, -2, -3, and -4. RESULTS: Our findings demonstrate that each of the five MMP genes studied were expressed in essentially all the tumors, while the adjacent marginal tissue samples showed a more varied picture: while stromelysin-3 was located to a majority of the marginal samples, matrilysin was expressed in four of seven adjacent samples, stromelysin-1 and MTI-MMP genes were each expressed in three of these samples, and stromelysin-2 transcript was only expressed in two marginal tissue samples. Whereas TIMP-1 and TIMP-2 transcripts were identified in all tumor and adjacent tissue samples studied, TIMP-3 was expressed, albeit often at low levels, in 17 of 20 tumor samples but only in three of seven adjacent tissues. The novel TIMP-4 gene was not expressed at all. CONCLUSIONS: Specific MMP (MMP-3, -7, -10, -14) and TIMP-3 transcripts observed in head and neck squamous cell carcinomas compared to their frequency in specimens of matching tissues provide important information about expression of extracellular matrix degrading enzymes and their tissue inhibitors in head and neck carcinomas.

Insulin-like growth factor family in malignant haemangiopericytomas: the expression and role of insulin-like growth factor I receptor.

Haemangiopericytoma is a rare soft tissue tumour originating from the contractile pericapillary cells. Relatively little is known about its molecular pathogenesis. To address this issue, the insulin-like growth factor family (IGFs) was analysed in 19 tumours collected from a human tumour bank network. Seven of the tumours were associated with severe hypoglycaemia. Of these, six were retroperitoneal and one was located in the leg. 3 out of the 19 tumours (15.8 per cent) were positive for insulin-like growth factor I (IGF I) mRNA and 11 were positive for IGF II mRNA (57.9 per cent). Almost 90 per cent of haemangiopericytomas expressed IGF I receptor (IGF IR) mRNA (17 out of 19), five (26.3 per cent) expressed IGF binding protein 1 (IGF BP1), three (15.8 per cent) expressed IGF BP2, and four (21 per cent) exhibited IGF BP3 mRNA. All of the 14 haemangiopericytomas examined with regard to specific receptor binding were IGF IR positive, ranging from 1.2 to 16.2 per cent. Binding was much higher in IGF I/IGF IR positive tumours (15.3+/-0. 7) than in IGF I negative/IGF IR positive tumours (5.1+/-3.3). The potential role of IGF IR as a growth promoting factor in malignant haemangiopericytoma was studied using antisense oligonucleotides and monoclonal antibody alphaIR3 that specifically inhibit IGF IR synthesis or activity. 10 microM IGF IR antisense oligonucleotides significantly inhibited the growth of haemangiopericytoma cells in culture, by around 50 per cent; monoclonal antibody against IGF IR (alphaIR3) also significantly inhibited proliferation. The data suggest that IGF IR may play an important role in the genesis and progression of malignant haemangiopericytomas.

Cervical metastases of head and neck squamous cell carcinoma correlate with loss of heterozygosity on chromosome 16q.

To assess the potential involvement of putative tumor suppressors or metastasis suppressors on chromosome 16q in head and neck squamous cell carcinoma (HNSCC), we have examined 42 primary HNSCCs for loss of heterozygosity (LOH) at 16q and correlated these findings with the occurrence of cervical nodal metastases and other clinical parameters. Seven of the 42 (17%) HNSCCs examined displayed LOH at chromosome 16q24. Three of the seven HNSCCs showed LOH at all of the informative loci analyzed along the chromosome arm, whereas the other four showed only loss of a subset of markers. When LOH at 16q was correlated with clinical parameters, there was no significant correlation with age, sex, clinical stage, T stage, N stage or survival. However, there was a correlation between LOH at chromosome 16q24 and involvement of cervical lymph nodes. Of the seven HNSCCs that had lost heterozygosity at 16q24, six had local metastases to lymph nodes indicating that LOH at 16q24 may have predictive value for the metastatic potential of HNSCCs.

p53 immunostaining of surgical margins as a predictor of local recurrence in squamous cell carcinoma of the oral cavity and oropharynx.

Local and regional recurrence is the principal reason for treatment failure in squamous cell carcinoma (SCC) of the head and neck. The conventional method of evaluating surgical margins for cellular atypia does not always predict risk of local recurrence accurately. Immunostaining of surgical margins for tumor markers may provide a more precise evaluation of risk of local recurrence. Paraffin-embedded tissue blocks of surgical margins from 24 patients with oral cavity and oropharyngeal squamous cell carcinoma were immunostained for p53 protein. Fifty-eight percent of the patients had at least one margin stain positive for p53, including eight of ten patients whose SCC recurred locally. The sample odds ratio test predicted a 5.333 times higher chance of local recurrence with at least one p53 positive surgical margin. The implications of these results for patient management and further investigations will be discussed.

Prognostic indicators for squamous cell carcinoma of the oral cavity: a clinicopathologic correlation.

Fifty-three patients with T1 squamous cell cancer of the floor of mouth and ventral surface of the tongue with a known clinical outcome were retrospectively analyzed and arbitrarily divided into "aggressive" and "nonaggressive" groups based on their clinical behavior. Various host and tumor factors were then evaluated in an attempt to determine whether the tumor behavior could have been predicted. The paraffin-embedded tumor specimens were evaluated for tumor differentiation, tumor thickness and tumor invasion, microvessel density, and p53 expression. In addition, a composite morphologic grading score was obtained by combining cell differentiation, nuclear polymorphism, mitosis activity, depth of infiltration, type of infiltration, and lymphatic infiltration. No single technique appeared capable of identifying "aggressive" behavior, although possibly an evaluation of composite factors might show promise in the future.

An HSVtk-mediated local and distant antitumor bystander effect in tumors of head and neck origin in athymic mice.

The "bystander effect," produced by ganciclovir-mediated killing of cells transduced with a herpes simplex virus thymidine kinase (HSVtk) gene, defines the cooperative killing of non-HSVtk-transduced cells. In vitro, a major contributor to this phenomenon is metabolic cooperation involving transfer of cytotoxic small molecules between cells via gap junctions. In this study, the bystander effect was assessed in vivo using cells of oral squamous cell carcinoma origin. Mixtures of HSVtk+ and HSVtk- tumor cells were implanted subcutaneously in the left flank of nude mice, and naive HSVtk- cells were implanted subcutaneously in the right flank. When tumors attained a size of 0.5 to 1 cm, the animals were treated with ganciclovir on a daily basis. The tumors comprised of mixed cells in the left flank resolved, consistent with a predicted bystander effect. The naive tumors in the right flank either resolved or became cytostatic showing little further growth compared to controls. Similar results were obtained when naive tumors were grown in both flanks and the tumor in the left flank received intratumoral injection of HSVtk retroviral producer cells or PA317 (HSVtk+) packaging cells, but not parental NIH 3T3 cells. Concomitant treatment with dexamethasone impaired the antitumor effect on the contralateral side. When these experiments were performed in SCID-Beige mice, there was a reduced antitumor effect on the ipsilateral flank and no antitumor response in the contralateral flank. Together with histology of regressing tumors, which showed an infiltration of lymphoid cells, these results are suggestive of an immune-related antitumor response that could account for the distant bystander effect.

The role of p53 tumor suppressor gene in human head and neck tumorigenesis.

Molecular genetics has led to new insights into diagnosis and treatment of human cancer. The alterations of tumor suppressor genes like retinoblastoma, p53 and others may have an important role in tumorigenesis. Mutations of p53 have been found in a majority of human malignancies including head and neck cancer. The distribution of p53 is different between types of tumors, suggesting environmental exposure as a cause active factor. The p53 mutation in head and neck tumors is an early event and appears to have a hot spot region at codons 238-248. While mutation and loss of heterozygosity at p53 are important in the genesis of head and neck cancer, other mechanisms such as binding of viral and cellular proteins to p53 are also likely to play a role.

Overexpression and amplification of glutathione S-transferase pi gene in head and neck squamous cell carcinomas.

Human glutathione S-transferase pi (GST-pi) may serve as a useful tumor marker because of the high frequency with which it is found in elevated levels in several tumor types. To determine whether GST-pi is useful as an indicator for cancers of the head and neck, expression of GST-pi mRNA was investigated by Northern analysis in this tumor type. Overexpression of GST-pi mRNA was detected in 9 of 36 (25%) primary head and neck squamous cell carcinomas (HNSCCs). When Southern blot analysis was used to examine the relationship between overexpression and amplification of the GST-pi gene, only 3 of 36 tumors (8%) showed GST-pi gene amplification. Thus, gene amplification is not critical to GST-pi mRNA overexpression in HNSCCs. Moderately and poorly differentiated HNSCCs tended to manifest elevated GST-pi mRNA compared with well differentiated tumors (30% for moderately and poorly differentiated tumors versus none of the well differentiated tumors examined). However, there was no significant correlation between GST-% mRNA overexpression and clinical stage, T stage (tumor size), N stage (neck nodal status), pathological nodes, or patient survival.

Prostaglandin H synthase isoenzyme distribution in the gingival tissue of patients with periodontitis: pronounced expression adjacent to gram-positive bacteria.

Prostaglandin (PGE(2)) is an inflammatory mediator that plays a critical role in the pathogenesis of periodontal disease. Prostaglandin H synthase (PGHS) a rate-limiting enzyme in PGE(2) biosynthesis exists as two separate isoforms (PGHS-1 and PGHS-2). We have previously demonstrated that both isoforms are generally present in the gingival tissue of periodontitis patients. This study explores in greater detail the variable distribution of each isoenzyme in both inflamed and non-inflamed gingival tissues of patients with periodontitis, and the relationship to adjacent bacteria. Although the positive staining for PGHS-1 was never as intense as for PGHS-2 in the same tissue specimen, either in inflamed or non-inflamed tissues, there was strong staining for both isoenzymes in the epithelium. The keratin layer did not stain. Non-keratinizing crevicular and junctional epithelium contained both isoenzymes through their full thickness in both inflamed and non-inflamed tissues. Pronounced staining of PGHS-2 was evident in the epithelia adjacent to Gram-positively stained organisms. In non-inflamed tissue, PGHS-1 and PGHS-2 were particularly evident in the spinous cell layer; however, fewer of the fibroblasts, endothelial cells, and resident mononuclear inflammatory cells stained positively for PGHS-1 as compared to PGHS-2, but this was less apparent in the inflamed tissues. The immunohistochemical staining patterns indicate that both crevicular and gingival epithelium are important sources of prostaglandin production in the gingival tissue of patients with periodontitis and that bacteria entrapped near to these sites may be important in promoting expression of inducible PGHS-2.

Overexpression of glutathione S-transferase pi messenger RNA and its relationship to gene amplification in head and neck squamous cell carcinoma.

Human glutathione S-transferase pi has been known to be a good marker for several tumor types because of the high frequency with which it is overexpressed. In order to determine whether GST pi is useful as an indicator for head and neck cancers, expression of GST pi was investigated by Northern analysis. Overexpression of mRNA was detected in 9 of 36 primary head and neck squamous cell carcinomas. To examine the relationship between overexpression and amplification of GST pi gene, Southern analysis was performed on all samples. Only 3 of the 36 tumors showed amplification GST pi genes, indicating that gene amplification may not play a key role in GST pi mRNA overexpression in these cancers.

The loss of heterozygosity in retinoblastoma and p53 suppressor genes as a prognostic indicator for head and neck cancer.

Inactivation of tumor suppressor genes, including p53 and retinoblastoma (Rb), are commonly found in all cancers, including head and neck squamous cell carcinoma. Alterations at either p53 or Rb, however, are only weakly associated with tumor aggressiveness. In many cancers loss of heterozygosity (LOH) at multiple loci is associated with decreased survival. The polymerase chain reaction and highly informative microsatellite markers were used to compare DNA from matched sets of 63 head and neck squamous cell cancers and normal tissue for LOH at the p53 and Rb loci. At p53, 50 were informative, with LOH occurring in 19 (38%). Of the 57 that were informative at Rb, LOH occurred in 21 (37%). Of the 46 that were informative at both p53 and Rb, LOH occurred in 10 (22%) at both loci. When LOH for p53 and Rb individually was compared to stage, differentiation, and survival, there was no correlation. However, the patients with LOH at both loci had a significantly poorer survival (P = .009). This strongly supports the contention that simultaneous alterations of these two tumor suppressor genes favor tumor aggressiveness and can be used as a prognostic indicator.

Absence of retinoblastoma gene product in human primary oral cavity carcinomas.

Oral cavity cancer is a major health concern worldwide. Despite advances in surgery, radiotherapy and chemotherapy over the past 35 years, there has been no significant enhancement in the survival of oral cavity cancer patients. Improved survival will require identification of reliable prognostic markers that provide a rational basis for assessment of risk for progression. The altered retinoblastoma (RB) gene has been linked to the hereditary retinoblastoma. This gene is defective in several types of human malignancies. The intent of this study was to evaluate the role of the RB gene in oral cavity tumorigenesis and to explore whether or not there is a relationship between the loss of RB protein and each of several clinicopathological parameters in oral cavity carcinomas. We have analysed the expression of the RB gene in four cell lines (J82, ML1, SaOS2 and WERI-RB-1), 182 oral cavity carcinomas (75 T1 and 107 T3 and T4 lesions) and 55 normal tissues adjacent to cancer by means of an immunohistochemical method and Western immunoblotting. The expression of RB protein was then correlated with clinical outcome in the patients with primary tumours. The significantly higher rate of altered RB expression was found in advanced oral cavity tumours (40 of 107; 37%) in comparison with low grade tumours (9 of 75; 7%). In T3 and T4 tumours, RB gene expression did not correlate with presence or absence of lymph node metastasis, degree of differentiation and patient survival. However, in the T1 cohort, poorer survival rate was seen for those patients who had a tumour with loss of RB protein. This study suggests that tumours in which the RB protein was altered were more aggressive than tumours in which the RB protein was present and that loss of RB protein in oral cavity cancer may be a prognostic variable of tumour progression.

Methods to detect P-glycoprotein-associated multidrug resistance in patients' tumors: consensus recommendations.

Multidrug resistance (MDR), especially that associated with overexpression of MDR1 and its product, P-glycoprotein (Pgp), is thought to play a role in the outcome of therapy for some human tumors; however, a consensus conclusion has been difficult to reach, owing to the variable results published by different laboratories. Many factors appear to influence the detection of Pgp in clinical specimens, including its low and heterogeneous expression; conflicting definitions of detection end points; differences in methods of sample preparation, fixation, and analysis; use of immunological reagents with variable Pgp specificity and avidity and with different recognition epitopes; use of secondary reagents and chromogens; and differences in clinical end points. Also, mechanisms other than Pgp overexpression may contribute to clinical MDR. The combined effect of these factors is clearly important, especially among tumors with low expression of Pgp. Thus, a workshop was organized in Memphis, Tennessee, to promote the standardization of approaches to MDR1 and Pgp detection in clinical specimens. The 15 North American and European institutions that agreed to participate conducted three preworkshop trials with well-characterized MDR myeloma and carcinoma cell lines that expressed increasing amounts of Pgp. The intent was to establish standard materials and methods for a fourth trial, assays of Pgp and MDR1 in clinical specimens. The general conclusions emerging from these efforts led to a number of recommendations for future studies: (a) although detection of Pgp and MDR1 is at present likely to be more reliable in leukemias and lymphomas than in solid tumors, accurate measurement of low levels of Pgp expression under most conditions remains an elusive goal; (b) tissue-specific controls, antibody controls, and standardized MDR cell lines are essential for calibrating any detection method and for subsequent analyses of clinical samples; (c) use of two or more vendor-standardized anti-Pgp antibody reagents that recognize different epitopes improves the reliability of immunological detection of Pgp; (d) sample fixation and antigen preservation must be carefully controlled; (e) multiparameter analysis is useful in clinical assays of MDR1/Pgp expression; (f) immunostaining data are best reported as staining intensity and the percentage of positive cells; and (g) arbitrary minimal cutoff points for analysis compromise the reliability of conclusions. The recommendations made by workshop participants should enhance the quality of research on the role of Pgp in clinical MDR development and provide a paradigm for investigations of other drug resistance-associated proteins.

Tumor angiogenesis in T1 oral cavity squamous cell carcinoma: role in predicting tumor aggressiveness.

BACKGROUND: Angiogenesis is necessary for tumor growth and metastasis. In breast and other cancers angiogenesis has been shown to correlate with tumor size, metastatic potential, and prognosis. Some studies of head and neck cancer have shown a similar correlation, although results are inconclusive. This study was performed to determine whether tumor angiogenesis can be used as a prognostic indicator for early oral cancers. METHODS: CD-31 immunostaining, the technique of choice for determining microvessel density, was utilized to investigate T1 squamous cell carcinomas of the ventral tongue and floor of the mouth. RESULTS: Adequate staining was achieved in 19 tumors. Seven tumors were deemed aggressive due to either the development of metastases or recurrence. The mean microvessel density in the aggressive patients was 43.1/hpf (range 15--79) and in the nonaggressive patients was 38.6/hpf (range 17--78). Statistical analysis failed to reveal any correlation between tumor aggressiveness and tumor angiogenesis in these early tumors. CONCLUSIONS: Tumor angiogenesis failed to predict tumor aggressiveness in T1 oral cavity carcinoma; however, low levels of neoangiogenesis were seen in all cases. For this reason this technique may prove more valuable in more advanced cancers.

HSV-tk gene therapy in head and neck squamous cell carcinoma. Enhancement by the local and distant bystander effect.

OBJECTIVES: To determine whether the bystander effect demonstrated in vitro for ganciclovir-mediated killing of a herpes simplex virus thymidine kinase (HSV-tk) gene-infected human squamous cell carcinoma is operative in vivo in a nude mouse model. DESIGN: Prospective study in a murine model. INTERVENTION: Human head and neck squamous cell carcinoma tumors were grown as xenografts on the flanks of 20 nude mice. The tumors in the left flank were then infected with the HSV-tk gene. Then, after 48 hours, the animals were treated with intraperitoneal ganciclovir twice daily. Assessment of the tumors on both flanks was performed over a 31-day period. MAIN OUTCOME MEASURES: Resolution of tumors infected with HSV-tk gene in animals treated with ganciclovir; resolution of tumors uninfected with HSV-tk gene on the contralateral flank in animals treated with ganciclovir. RESULTS: Following HSV-tk gene therapy in nude mice, complete resolution of HSV-tk-gene-infected human head and neck squamous cell carcinoma tumors was observed following ganciclovir treatment. Uninfected tumors were also noted to regress, but not completely resolve, in response to intraperitoneal ganciclovir (distant bystander effect). CONCLUSIONS: This study confirms that the local and distant bystander effects exist in this murine model, enhancing the possibility of its role for treatment of human squamous cell carcinoma of the head and neck.

Molecular genetics of malignant insulinoma.

Malignant insulinoma is an rare form of cancer with poor prognosis and a reported 5-year survival of 35%. Relatively little is known about the etiology of this disease or of the oncogenes and tumor suppressor genes that participate in its genesis and progression. To address this issue, several protooncogenes, including K-ras, N-ras, erbB-2, erbB-3,c-myc, c-fos, c-jun were examined. Also analyzed was the expression of the growth factors TGF-alpha, EGF, and insulin as well as the EGF receptor (EGF-R), p53 and the putative anti-metastasis gene nm23-H1. These were examined in malignant insulinomas, benign insulinomas, pancreatic B cell hyperplasias and in normal endocrine pancreas. Normal endocrine pancreas showed moderate immunoreaction for c-myc and a strong reaction for insulin. All other parameters were negative. Benign pancreatic B cell hyperplasias were slightly or moderately positive for N-ras and TGF-alpha, and were weakly positive for EGF-R. They were strongly positive for c-myc and insulin. In malignant insulinomas there was strong immunoreaction for c-myc, TGF-alpha, N-ras, K-ras and p53. Insulin reaction was moderate or strong. Molecular genetic studies have been performed for the presence of activating point mutations in codon 12 of the c-K-ras oncogene. Mutations were detected using primer-mediated, mutant-enriched, polymerase chain reaction-restriction fragment length polymorphism analysis and were further characterized by allele-specific oligonucleotide hybridization. Four out of six patients with malignant insulinoma and two out of eight patients with benign insulinoma harbored K-ras point mutations at codon 12. All patients with mutated K-ras oncogene also had elevated levels of p53 protein as well as c-myc and TGF-alpha. In one extremely malignant case we found concomitant mutation at codon 12 of K-ras and codon 61 of the N-ras gene. Our data are consistent with the idea that malignant progression is accompanied by the progressive accumulation of multiple genetic lesions and suggest that activation of myc, TGF-alpha and ras genes may be early events in the development of insulinoma.

Effect of Wnt-1 antisense RNA on the outgrowth of a mammary adenocarcinoma cell line expressing that oncogene.

Aims-To investigate the effect of Wnt-1 antisense RNA on the outgrowth of a mammary tumour cell line expressing that oncogene.Methods-A plasmid (pMT 70), containing Wnt-1 cDNA, was cut with appropriate enzymes and inserted into a eukaryotic expression vector (pMAMneo). A mammary tumour cell line (CAC-L153) was transfected with the expression vector and cells with the vector in sense and antisense orientation were selected.Results-Tumour cells with the expression vector in the antisense orientation had a notable reduction in expression of Wnt-1 protein and a considerable reduction in tumour outgrowth compared with controls.Conclusions-The results indicate that the Wnt-1 proto-oncogene may be a possible target for antisense therapy.

Stage of disease confounds apparent relationship between levels of N-ras and duration of survival in head and neck tumours.

The objective of this study was to determine whether elevated levels of N-ras correlated with clinicopathological data. Complete clinical data were available on 133 of 481 patients surgically treated for squamous cell carcinoma of the head and neck (SCCHN) who had immunohistochemical data for N-ras. Advanced stages of disease were strongly related to the staining for N-ras in tumour cells (P = 0.0031). The stage of disease was inversely related to duration of survival (P = 0.0017). Initial statistical evaluation revealed an apparent correlation between survival and N-ras staining. However, duration was found to be independent of the level of N-ras. The illusory relationship initially was a result of the confounding effect of the stage of disease.

Amplification and overexpression of the cyclin D1 gene in head and neck squamous cell carcinoma.

Aims-To determine cyclin D1 gene amplification and expression levels in head and neck squamous cell carcinoma (HNSCC) patients.Methods-Total RNA and genomic DNA were isolated from 40 samples of HNSCC tissue and matched normal tissue and were hybridised with a cyclin D1 cDNA probe. Northern and Southern analyses were used to detect mRNA overexpression and cyclin D1 gene amplification, respectively.Results-15 of the 40 HNSCC samples examined (38%) showed cyclin D1 gene amplification. Of these 15 samples, all 13 from which RNA was available showed increased cyclin D1 expression.Conclusions-HNSCC patients with both amplification and overexpression of the cyclin D1 gene are at greater risk than patients who showed no cyclin D1 gene amplification; amplification and over-expression of the cyclin D1 gene may play an important role in the progression of HNSCC and in clinical outcome.