Prognostic value of cathepsin L and its inhibitor headpin in oral squamous cell carcinoma.

OBJECTIVE: To investigate the clinicopathological and prognostic significance of the expression of cathepsin L and its inhibitor headpin, in oral squamous cell carcinoma. DESIGN: Immunohistochemical studies were performed on 56 oral squamous cell carcinoma samples. We evaluated the relationship between cathepsin L and headpin expression versus patients' clinicopathological factors and survival. RESULTS: The group that was positive for cathepsin L expression tended to have positive metastatic neck lymph nodes and a poorer prognosis. Headpin expression was not related to metastasis or prognosis. Well differentiated squamous cell carcinoma had higher levels of headpin expression compared with poorly differentiated squamous cell carcinoma. CONCLUSION: Cathepsin L expression is related to the invasive and metastatic potential of oral squamous cell carcinoma.

Parotid gland oncocytosis: CT findings with histopathologic correlation.

Oncocytic neoplasms result from metabolically altered cells that accumulate abundant mitochondria within their cytoplasm by oncocytic metaplasia. In this report, the CT findings are described and correlated with the histopathologic features of a case of oncocytoma involving the parotid gland that arose in a background of nodular oncocytic hyperplasia. When imaging demonstrates multiple small nodules in the parotid gland with a large, solid, or cystic mass, the diagnosis of oncocytic neoplasia should be considered.

Human LBP-32/MGR is a repressor of the P450scc in human choriocarcinoma cell line JEG-3.

Steroid hormones regulate a wide range of physiologic functions in humans. The cholesterol side-chain cleavage enzyme P450scc regulates the initial step of biosynthesis of all steroid hormones. We investigated the expression of P450scc by studying a potential regulator of P450scc, LBP-32/MGR. Using a Northern blot, we found that LBP-32/MGR mRNA was expressed mainly in the human placenta. Using radiation hybrid mapping, we identified LBP-32/MGR on human chromosome 2p25. Recombinant LBP-32/MGR protein bound preferentially to a DNA fragment from the promoter of P450scc in vitro and exhibited clear nuclear localization in transfected cells. Luciferase reporter gene assays showed that LBP-32/MGR specifically repressed transcriptional activation of the human P450scc promoter. Because placental P450scc expression is essential for pregnancy and steroid biosynthesis, the placental expression and transcriptional repressor activity of LBP-32/MGR in JEG-3 cells suggest it has a role as a transcriptional modulator of steroid biosynthesis.

Genetic heterogeneity in saliva from patients with oral squamous carcinomas: implications in molecular diagnosis and screening.

We performed microsatellite analysis at chromosomal regions frequently altered in head and neck squamous carcinoma on matched saliva and tumor samples from 37 patients who had oral squamous carcinoma. The results were correlated with the cytologic findings and traditional clinicopathologic factors to assess the diagnostic and biological potential of these markers. Our data showed that 18 (49%) of the saliva samples and 32 (86%) of the tumors had loss of heterozygosity (LOH) in at least one of the 25 markers studied. In saliva, the combination of markers D3S1234, D9S156, and D17S799 identified 13 (72.2%) of the 18 patients with LOH in saliva (P < 0.001). For tumors, markers D3S1234, D8S254, and D9S171 together identified 27 (84.3%) of the 32 tumors with LOH at any of the loci tested (P < 0.001). Eleven (55%) of the 20 saliva samples with cytologic atypia and seven (35%) of the 17 specimens without atypia had LOH. Significant correlation between LOH in tumor at certain markers and smoking and alcohol use was found. Our results indicate that: 1) epithelial cells in saliva from patients with head and neck squamous tumorigenesis provide suitable material for genetic analysis; 2) combined application of certain markers improves the detection of genetic alteration in these patients; 3) clonal heterogeneity between saliva and matching tumor supports genetic instability of the mucosal field in some of these patients; and 4) LOH at certain chromosomal loci appears to be associated with smoking and alcohol consumption.

All-trans-retinoic acid enhances the effect of adenovirus-mediated wild-type p53 gene transfer in head and neck squamous cell carcinoma.

OBJECTIVES: Adenovirus-mediated p53 (AdCMVp53) gene therapy for cancer is currently undergoing phase III clinical trials. One problematic aspect of this therapy is that the current protocols result in low transduction of the therapeutic virus in vivo. To search new modalities that can enhance the effect of AdCMVp53 gene therapy, we focused on retinoids. METHODS: To study the effect of ATRA in combination with AdCMVp53 gene therapy, we pretreated head and neck squamous cell carcinoma (HNSCC) cells for 72 hours with a low-dose All-trans-retinoic acid (ATRA) (10-7 M-10-8 M) which will not affect the in vitro cell growth, and then infected the cells with low MOI (30MOI) AdCMVp53. In vitro cell proliferation assays, cell cycle assays were performed. Expression of p53 and p53-related gene products, BAX and p21, were examined. RESULTS: The combined treatment with ATRA and Ad-p53 suppressed cell growth and induced apoptosis significantly more than AdCMVp53 treatment alone (P <.05). p53 expression significantly increased more after the combined treatment than after either treatment alone, at both the transcription and protein levels. In addition, increased expression of p21 and BAX, which are downstream gene products of p53, was observed in the combination. ATRA also enhanced the expression of green fluorescent protein (GFP) transduced by an adenovirus-cytomegalovirus (CMV)-GFP vector suggesting ATRA enhances adenovirus-CMV-promoted vectors through transcription. CONCLUSIONS: Our results indicate that ATRA enhances AdCMVp53 expression through transcriptional mechanisms and can synergistically induce apoptosis in HNSCC cells. ATRA has a potential to enhance the effect of adenovirus-mediated p53 gene therapy for HNSCC.

Expression of protease-activated receptor 1 in oral squamous cell carcinoma.

Protease-activated receptor 1 (PAR-1) is a G-coupled membrane protein. In this study, we analyzed the expression of PAR-1 in oral squamous cell carcinomas (SCCs). PAR-1 was expressed in oral SCCs, but the level of PAR-1 protein was lower in non-metastatic cells than in metastatic cells. Thrombin stimulated the growth of metastatic cells, and both thrombin and thrombin receptor activation peptide (TRP) enhanced the adhesion of these cells to fibronectin, but had no effect on non-metastatic cells. Thrombin and TRP also induced matrix metalloproteinase (MMP)-2 and MMP-9 activities in metastatic cells. These results suggest that PAR-1 may contribute to the growth and invasive potential of oral SCC.

Combined interferon-alfa, 13-cis-retinoic acid, and alpha-tocopherol in locally advanced head and neck squamous cell carcinoma: novel bioadjuvant phase II trial.

PURPOSE: Retinoids and interferons (IFNs) have single-agent and synergistic combined effects in modulating cell proliferation, differentiation, and apoptosis in vitro and clinical activity in vivo in the head and neck and other sites. Alpha-tocopherol has chemopreventive activity in the head and neck and may decrease 13-cis-retinoic acid (13-cRA) toxicity. We designed the present phase II adjuvant trial to prevent recurrence or second primary tumors (SPTs) using 13-cRA, IFN-alpha, and alpha-tocopherol in locally advanced-stage head and neck cancer. PATIENTS AND METHODS: After definitive local treatment with surgery, radiotherapy, or both, patients with locally advanced SCCHN were treated with 13-cRA (50 mg/m(2)/d, orally, daily), IFN-alpha (3 x 10(6) IU/m(2), subcutaneous injection, three times a week), and alpha-tocopherol (1,200 IU/d, orally, daily) for 12 months, with a dose modification. Screening for recurrence or SPTs was performed every 3 months. RESULTS: Tumors of 11 (24%) of the 45 treated patients were stage III, and 34 (76%) were stage IV. Thirty-eight (86%) of 44 patients completed the full 12-month treatment (doses modified as needed). Toxicity generally was consistent with previous IFN and 13-cRA reports and included mild to moderate mucocutaneous and flu-like symptoms; occasional significant fatigue (grade 3 in 7% of patients), mild to moderate hypertriglyceridemia in 30% of patients who continued treatment along with antilipid therapy, and mild hematologic side effects. Six patients did not complete the planned treatment because of intolerable toxicity or social problems. At a median 24-months of follow-up, our clinical end point rates were 9% for local/regional recurrence (four patients), 5% for local/regional recurrence and distant metastases (two patients), and 2% for SPT (one patient), which was acute promyelocytic leukemia (ie, not of the upper aerodigestive tract). Median 1- and 2-year rates of overall survival were 98% and 91%, respectively, and of disease-free survival were 91% and 84%, respectively. CONCLUSION: The novel biologic agent combination of IFN-alpha, 13-cRA, and alpha-tocopherol was generally well tolerated and promising as adjuvant therapy for locally advanced squamous cell carcinoma of the head and neck. We are currently conducting a phase III randomized study of this combination (v no treatment) to confirm these phase II study results.

Epidermal growth factor receptor-targeted therapy with C225 and cisplatin in patients with head and neck cancer.

C225, a human-mouse chimerized monoclonal antibody directed against the epidermal growth factor receptor (EGFr), has a synergistic effect with cisplatin in xenograft models. To determine the tumor EGFr saturation dose with C225 and the fate of infused C225, we conducted a Phase Ib study with C225 in combination with cisplatin in patients with recurrent squamous cell carcinoma of the head and neck. Using tumor samples, we assessed tumor EGFr saturation by antibody using immunohistochemistry studies, the EGFr tyrosine kinase assay, and detection of the EGFr/C225 complex formation by immunoblot. Potential candidates were screened for EGFr expression in their tumors, and 12 patients who had high levels of EGFr expression and tumors easily accessible for repeated biopsies (pretherapy, 24 h after first C225 infusion, 24 h before third C225 infusion) were entered at three different dose levels of C225 with a fixed dose of cisplatin. The median value of tumor EGFr saturation increased to 95% at the higher dose levels. EGFr tyrosine kinase activity was significantly reduced after C225 infusion, and EGFr/C225 complexes were also detected at higher doses of C225. The loading dose of C225 at 400 mg/m(2) with a maintenance dose at 250 mg/m(2) achieved a high percentage of saturation of EGFr in tumor tissue, and these doses were recommended for Phases II or III clinical trials. Six (67%) of nine evaluable patients achieved major responses, including two (22%) complete responses. Mild to moderate degrees of allergic reaction and folliculitis-like skin reactions were demonstrated. We conclude that infused C225 binds and significantly saturates tumor EGFr, which may render a high degree of antitumor activity, and provides a novel mechanism for targeting cancer therapy for patients who have EGFr expression in their tumors.

Evidence that the death receptor DR4 is a DNA damage-inducible, p53-regulated gene.

DR4 (TRAIL-R1), a member of the tumor necrosis factor receptor superfamily, is a cell surface receptor that triggers the apoptotic machinery upon binding to its ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Although three other TRAIL receptors DR5, DcR1, and DcR2 are induced by DNA damage and are regulated by the wild-type p53 tumor suppressor, it was not known whether these factors also affect DR4 expression. In this study, we found that DR4 expression is also enhanced by DNA damage whether induced by ionizing radiation or by chemotherapeutic agents. The induction was observed predominantly in cells containing wild-type p53 and was similar to the regulation patterns of DR5 and Fas, two other members of the family which are known to be regulated by p53. Transfection of HPV 16 E6 gene into cells with wild-type p53, which decreased the level of p53 protein, resulted in suppression of DR4 induction by DNA-damaging agents. Conversely, introduction of exogenous wild-type p53 through adenovirus infection has led to upregulation of endogenous DR4 in cells with mutant p53. Moreover, the transcription inhibitor actinomycin D abolished DNA-damaging agent-induced DR4 expression. Thus, DR4 appears to be a DNA damage-inducible, p53-regulated gene.

SCCA1 expression in T-lymphocytes peripheral to cancer cells is associated with the elevation of serum SCC antigen in squamous cell carcinoma of the tongue.

Squamous cell carcinoma (SCC) antigen has been used for the management of SCC arising in various cites including head and neck region. However, the true mechanism of the elevation of this protein in the serum of patients with SCC is still unknown. SCC antigen belongs to the superfamily of serine protease inhibitors. Recently, molecular studies show that serum SCC antigen is transcribed by two nearly identical genes (SCCA1 and SCCA2), and is mainly produced by SCCA1. The objective of this study is to clarify the mechanism of the elevation of SCC antigen in oral tongue SCC patients and to identify cells histologically, which are responsible for serum SCC antigen production. In this study, we examined SCCA1 expression in a series of four head and neck SCC (HNSCC) cell lines, and found that all expressed equal to low SCCA1 protein as compared with the normal human oral keratinocyte. Using the double immunohistochemical technique to examine the expression pattern of SCCA1 in 86 cases of oral tongue squamous cell carcinoma, SCCA1 immunostaining was observed in the cytoplasm of cancer cells and T-lymphocytes peripheral to cancer cells. We also compared the clinicopathological features including serum SCC antigen level of the oral tongue SCC cases with the immunohistochemical SCCA1 expression pattern, and found that elevated serum SCC antigen level was significantly correlated with SCCA1 expression not in cancer cells, but in T-lymphocytes peripheral to cancer cells. These results suggest that T-lymphocytes peripheral to cancer cells may be responsible for serum SCC antigen production in HNSCC patients.

Phase II study of paclitaxel, ifosfamide, and carboplatin in patients with recurrent or metastatic head and neck squamous cell carcinoma.

BACKGROUND: In the current study the authors assessed the antitumor activity (including response rate, duration of response, and survival) and toxicity profile (including anorexia, fatigue, emesis, and peripheral neuropathy) of a combination of paclitaxel, ifosfamide, and carboplatin (TIC) in patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN). The trial hypothesis was that the TIC therapeutic index would be as high as that of paclitaxel, ifosfamide, and cisplatin (TIP) in this setting, but with less toxicity. METHODS: Patients with recurrent or metastatic SCCHN were treated with 175 mg/m(2) of paclitaxel as a 3-hour infusion on Day 1, 1000 mg/m(2) of ifosfamide as a 2-hour infusion on Days 1-3, 600 mg/m(2) of mesna on Days 1-3, and carboplatin (area under the concentration-time curve of 6) as a 30-minute infusion on Day 1; the regimen was repeated every 3-4 weeks. All patients were premedicated with dexamethasone, diphenhydramine, and cimetidine before paclitaxel infusion. Prophylactic hematopoietic growth factors were not given. RESULTS: Among 56 patients entered onto the study, 55 patients were analyzed for survival rates (locoregional recurrence alone in 56% of patients and distant metastasis with or without locoregional recurrence in 44% of patients). Fifty-four patients were evaluable for tumor response and toxicity. A total of 32 patients (59%) had disease that responded to treatment; the complete response rate was 17% (9 of 54 patients). The median duration of the responses was 3.7 months (95% confidence interval [95% CI], 3.4-7.8 months) and that of complete responses was 9.7 months (95% CI, 7.4 months to date of last follow-up). The median duration of follow-up care in all patients was 13.5 months. The median survival time for all patients was 9.1 months (95% CI, 7.9-12.2 months). The regimen was well tolerated. Neutropenic fever developed in 30% of the patients; 1 patient died of neutropenia and sepsis. Other toxic effects included Grade 2-3 anorexia in 13% of patients, Grade 2-3 weight loss in 11% of patients, Grade 2-3 fatigue in 27% of patients, Grade 2-3 nausea/emesis in 13% of patients, and Grade 2-3 peripheral neuropathy in 9% of patients (toxicity grading based on the National Cancer Institute's Common Toxicity Criteria). Red blood cell and platelet transfusions were required in 13% and 7% of patients, respectively. CONCLUSIONS: The TIC regimen had high antitumor activity in patients with recurrent or metastatic SCCHN, with a 59% major response rate (17% complete response rate with relatively durable complete responses). Neutropenic fever developed in 30% of the patients, the incidence of which might have been decreased by prophylactic antibiotics or hematopoietic growth factor support. Other toxic effects included significantly lower rates and less severe instances of anorexia, emesis, fatigue, and peripheral neuropathy than those reported with the previously studied TIP regimen. The TIC regimen currently is being studied as an induction chemotherapy regimen in previously untreated patients with locally advanced SCCHN. The activity of TIC (a novel paclitaxel and ifosfamide-based regimen) in patients with recurrent or metastatic SCCHN should be confirmed in a Phase III randomized trial.

p73 gene alterations and expression in primary oral and laryngeal squamous carcinomas.

p73, a recently identified gene, maps to chromosome region 1p36.3, which is frequently deleted in a variety of solid tumors. Although the gene shares sequence and functional homologies with p53, its suppressor function has not been proven. We investigated for the first time the genetic and expression status of the p73 gene and analyzed its flanking microsatellite loci on chromosome 1p36.3 in 67 primary oral and laryngeal squamous cell carcinomas to determine their association with these tumors. Our results reveal two missense mutations at codons 469 and 477 and a silent mutation at codon 349 in the C-terminal domain. Site-directed mutagenesis of p73 cDNA with these mutations and a p21 transactivation assay failed to show any significant functional consequences of these mutations. Microsatellite analysis of the flanking loci of p73 in region 1p36 showed overall alterations (loss of heterozygosity and instability) frequency of 39%, 16% at the proximal marker and 46% at the distal markers. Of the 21 cases for which we did protein expression analyses, 11 tumors had a >2-fold variation compared with matching histologically normal mucosa. Our study shows that: (i) intragenic alterations in this gene are rare and lack functional significance; (ii) its variable expression argues against a tumor suppressor function; (iii) this gene plays a minor role in head and neck squamous carcinoma; (iv) a distal site to this gene on 1p36 may harbor another suppressor gene.

The role of neck dissection after chemoradiotherapy for oropharyngeal cancer with advanced nodal disease.

OBJECTIVE: To analyze and compare the effectiveness of sequential platinum-based chemotherapy and radiotherapy with and without selective neck dissection in patients with N2a and greater stage node-positive squamous cell carcinoma of the oropharynx. DESIGN: Nonrandomized controlled trial. SETTING: Tertiary referral center. PATIENTS: Sixty-six patients with squamous cell carcinoma of the oropharynx staged N2a or greater. INTERVENTIONS: Platinum-based induction chemotherapy followed by definitive radiation therapy; and selective neck dissections 6 to 10 weeks following the completion of radiation therapy in patients with radiographic evidence suggesting residual neck disease. MAIN OUTCOME MEASURES: Locoregional recurrence and disease-free survival. RESULTS: Of 66 patients, 24 (36%) had complete responses in the primary local tumor (oropharynx) and regional disease (neck nodes), as assessed clinically and radiographically. These patients had lower rates of locoregional recurrence than did patients showing no or partial responses, but the differences were not significant (P>.05). Of 18 patients undergoing neck dissection, 10 (56%) had pathological evidence of residual tumor. Patients showing a complete response of regional and neck disease had significantly improved disease-specific and overall survival (P = .01 for both) compared with patients showing no or partial responses of their neck disease. Patients with no or partial responses who underwent neck dissections had significantly improved overall survival compared with similar patients who did not undergo neck dissections (P = .002). CONCLUSIONS: Even in patients with bulky nodal disease, a complete response in the neck to sequential chemotherapy and radiotherapy may indicate that neck surgery is not necessary for good locoregional control and improved disease-free survival. Neck dissection is recommended for patients with no or partial radiographic responses.

Chemokines in cancer.

Chemokines are small, chemotactic cytokines that direct migration of leukocytes, activate inflammatory responses and participate in many other pleiotropic functions, including regulation of tumour growth. Chemokines modulate tumour behaviour by three important mechanisms: regulation of tumour-associated angiogenesis, activation of a host tumour-specific immunological response, and direct stimulation of tumour cell proliferation in an autocrine fashion. All of these mechanisms are promising points of cancer intervention, and preclinical mouse models suggest that chemokine antagonists and agonists could become important in the development of new anticancer therapies.

Isolation of a peptide for targeted drug delivery into human head and neck solid tumors.

Lack of tumor specificity remains a major problem with chemotherapies in that side effects prevent the delivery of dosages of drugs that are required to eliminate tumors. In this report, we describe the isolation of a 12-mer peptide (HN-1), with approximately 1% of the mass of typical antibodies, that meets several criteria for targeted drug delivery into a solid tumor. First, internalization of HN-1 by human head and neck squamous cell cancer (HNSCC) cells suggests that HN-1 is capable of translocating drugs across cell membranes. Second, HN-1 appears to be HNSCC-specific, given its reduced uptake by nonmalignant human oral keratinocytes and other types of human cells, its preferential binding to primary HNSCC, and its localization to HNSCC-derived xenografts. Third, the presence of HN-1 within HNSCC xenografts suggests that it is capable of penetrating tumor tissues. Our results establish the utility of tumor-specific peptides for targeted drug delivery into solid tumors.

Genomic cloning, mapping, structure and promoter analysis of HEADPIN, a serpin which is down-regulated in head and neck cancer cells.

Headpin is a novel serine proteinase inhibitor (serpin) that is down-regulated in squamous cell carcinoma of the oral cavity and in squamous cell carcinoma cell lines of the head and neck. Using a panel of 18q21.3 YAC clones, we mapped and cloned the HEADPIN gene. The gene spans 10 kb and is composed of eight exons and seven introns. The genomic structure is identical with some other ovalbumin serpins (ov-serpins) in terms of the numbers, position and phasing of the intron/exon boundaries. HEADPIN was mapped within the serpin cluster in 18q21.3 between MASPIN and SCCA2 as follows: cen-MASPIN-HEADPIN-SCCA2-SCCA1-tel. The transcription start site was determined and the promoter activity of the 5'-flanking region was analyzed. Luciferase promoter assays in HaCaT cells showed that the -432 to -144 nucleotide region has functional promoter activity. The activity of the promoter/enhancer was not observed in head and neck cancer cell lines TU167 and UMSCC1 which lack headpin expression. These data suggest that the differential expression of headpin in normal and carcinoma-derived cells is regulated at the transcriptional level. Understanding the genomic organization and transcriptional regulation of the ov-serpins clustered within 18q21. 3 provides a critical framework for assessing their potential role in cancer.

Expression of protein mediators of type I interferon signaling in human squamous cell carcinoma of the skin.

IFN-based therapy has been shown to be active in the treatment of squamous cell carcinoma (SCC) of the skin and has promise for chemoprevention and treatment of several other cancers. In an effort to better understand the molecular mechanism of this activity, we have determined the expression pattern of several of the protein mediators of type I IFN signaling by immunohistochemistry in cutaneous SCC, SCC metastases, and adjacent nonmalignant epithelium from patient biopsies. All of the proteins, signal transducer and activator of transcription (STAT) 1alpha/beta, STAT2, p48, STAT3a, and STAT3beta, are expressed at varying levels in the adjacent epidermis, as well as in other epidermal and dermal cell types. For the majority of samples tested, the expression of one or more of these proteins was reduced in SCC primary tumors compared with the adjacent nonmalignant epithelial cells, as determined by manual scoring. Quantitative densitometry of several samples revealed differences that are statistically significant. Our study provides the first direct evidence for the expression of the IFN-stimulated gene factor 3 (STAT1alpha/beta, STAT2, and p48) and STAT3alpha and STAT3beta mediators of IFN-alpha/beta signaling in human skin and skin-derived SCCs. These data have led to the hypothesis that the loss of IFN sensitivity may contribute to the development and progression of skin SCC.

The current status of gene therapy.

Locoregionally recurrent head and neck squamous cell carcinoma is a logical target for direct delivery of gene therapy approaches. Because the protein p53 plays a role in cell cycle regulation and in apoptosis, p53 gene transfer was initially tested in head and neck cancer patients by injecting the primary or regional tumor with an adenoviral vector possessing wild-type p53. Adenoviral p53 was demonstrated to be safe and well tolerated; furthermore, activity was observed. Several randomized studies of adenoviral p53 are now under way in patients with head and neck squamous cell carcinoma to determine its role as a surgical adjuvant in untreated disease and in combination with DNA-damaging agents.