Locally advanced mammary analogue secretory carcinoma of the parotid gland.

Mammary analogue secretory carcinoma (MASC) has recently been recognized as a salivary gland tumour that is characterized by the ETV6-NTRK3 fusion gene. A case of locally advanced MASC of the parotid gland in a 67-year-old man is presented here. The patient visited the hospital due to a large right infra-auricular mass, which had been enlarging gradually over a period of 2years. Contrast-enhanced computed tomography (CT) demonstrated a multilocular mass, 75×63mm in size, containing a fluid component with non-uniform contrast effects in the interior portion. The mass had invaded the orbit, skull base, and parapharyngeal space. The patient had neither lymph node nor distant metastasis. The tumour showed tubular and ductal proliferation lined by a single layer of neoplastic cuboidal cells with clear foamy cytoplasm. Characteristic hobnail cells were observed. Expression of ETV6-NTRK3 fusion transcript in the tumour tissues was confirmed by RT-PCR. The final diagnosis was MASC (T4bN0M0, stage IVB). The patient received cetuximab together with radiotherapy at a total dose of 66Gy. After treatment, CT showed a slightly reduced tumour volume, indicating stable disease. More than 56 months after treatment, the patient remains alive with no remarkable change in the tumour.

Double sentinel lymph node mapping with indocyanine green and 99m-technetium-tin colloid in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) frequently metastasizes to cervical lymph nodes, which is the most known prognostic factor. Screening methods to identify sentinel lymph nodes (SLNs) are therefore of great interest for the management of potential neck metastasis. The purpose of this study was to evaluate the clinical benefit of double SLN mapping with indocyanine green (ICG) and 99m-technetium-tin colloid ((99m)Tc-tin colloid) for sentinel node navigation surgery (SNNS). Between 2007 and 2010, 16 patients diagnosed with OSCC were investigated by SLN biopsy using the double mapping method. (99m)Tc-tin colloid was injected into the peri-tumoural region on the preoperative day, and ICG was administered intraoperatively in the same position to assist in detecting nodes during surgery. Based on the gamma-ray signal and near-infrared (NIR) fluorescence of ICG, SLNs were identified and thereafter assessed pathologically and genetically for cancer involvement. Radio-guided detection was successful for all patients. ICG mapping identified a relatively larger number of nodes, suggesting that several non-SLNs were potentially involved. The double mapping method assisted surgeons to explore SLNs. Since the ICG fluorescence was shielded by the subcutaneous fatty tissue and the muscle layer including platysma and sternocleidomastoid, it was necessary to retract the tissue away from nodes.

Friend leukaemia insertion (Fli)-1 is a prediction marker candidate for radiotherapy resistant oral squamous cell carcinoma.

Radiotherapy is commonly used to treat oral squamous cell carcinoma (OSCC), but its therapeutic effects are unpredictable. To determine which genes correlate with radiation resistance in oral cancer, the authors evaluated radiation sensitivity using a standard colony formation assay with a gene microarray system for seven OSCC cell lines. They found significant associations between dozens of gene-expression levels and radiation resistance of OSCC cell lines. Following analysis of the different radiosensitive cancer cell lines, the friend leukaemia insertion (Fli)-1 gene was selected as a prediction marker gene for OSCC radiotherapy resistance. Fli-1 expression was associated with radiation resistance in OSCC patients. These data help to predict the effects radiation therapy has on OSCC, in turn contributing to the development of alternative radiation therapies.

Mandibular osteoblastic metastasis of poorly differentiated carcinoma of the thyroid gland.

Metastasis of malignant tumors to the oral cavity is rare. The authors report a case of thyroid carcinoma with mandibular osteoblastic metastasis. An 83-year-old female presented with lower jaw swelling and pain. An elastic hard subcutaneous mass was observed in the median mandible. X-ray images confirmed a tumor lesion with periosteal reaction spreading radially from the mandible. A biopsy revealed nests of large, polygonal tumor cells growing in a supporting fibrovascular framework. The patient's anamnesis included thyroid carcinoma with lung metastasis, 2 years ago, treated by total enucleation of the thyroid and excision of the superior lobe of the left lung. Biopsy, primary and metastatic tumor samples all tested positive for thyroglobulin, suggesting a thyroid follicular epithelial origin. Mandibular metastasis of poorly differentiated carcinoma of the thyroid gland was diagnosed. Consent for further treatment was not obtained. The patient died 1 year and 7 months later.

Identification of homozygous deletions of tumor suppressor gene FAT in oral cancer using CGH-array.

Homozygous deletions (HD) provide an important resource for identifying the location of candidate tumor suppressor genes. To identify the tumor suppressor gene in oral cancer, we employed high-resolution comparative genomic hybridization (CGH)-array analysis. We identified a homozygous loss of FAT (4q35), a new member of the human cadherin superfamily, from genome-wide screening of copy number alterations in one primary oral cancer. This result was evaluated by genomic polymerase chain reaction in 13 oral cancer cell lines and 20 primary oral cancers and Southern blot in the cell lines. We found frequent exonic HD of FAT in the cell lines (3/13, 23%) and in primary oral cancers (16/20, 80%). FAT expression was absent in these cell lines. Homozygous deletion hot spots were observed in exon 1 (9/20, 45%) and exon 4 (7/20, 35%). Moreover, loss of gene expression was identified in other types of squamous cell carcinoma. The methylation status of the FAT CpG island in squamous cell carcinomas correlated negatively with its expression. Our results identify mutations in FAT as an important factor in the development of oral cancer and indicate the importance of FATs function in some squamous cell carcinomas.

Microvessel density and expression of vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived endothelial growth factor in oral squamous cell carcinomas.

Angiogenesis, the growth of capillary vessels, plays an important role in the metabolic functions of malignant tissues. Tumor growth and malignant transformation are considered to be dominated by uncontrolled angiogenesis. To understand the mechanism of increased vascularity associated with malignant tissues, we immunohistochemically evaluated microvessel density (MVD) and the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived endothelial growth factor (PDGF) in oral cancers. Microvessel density did not differ significantly between normal oral mucosa and epithelial dysplasia, but was significantly increased in tumor tissues. Expression of angiogenic factors was not found in normal oral mucosa, but increased in association with increasing vascularity in OSCC tissue. In tumor tissue, angiogenic factor expression correlated with MVD. MVD in OSCC was related to T stage, tumor differentiation, and stage of invasion. VEGF expression also correlated with tumor differentiation and the stage of invasion. These findings suggest that VEGF might play an important role in tumor angiogenesis of OSCC.

Malignant pleural mesothelioma metastasis to the mandible.

Malignant mesothelioma is a rare tumor arising from the pleura or peritoneum. Distant hematogenous metastasis is seen in more than half of cases, preferentially to the brain, lung, bone and soft tissues [Br. J. Dis. Chest 70 (1976) 246]. There has been only one previous report of this tumor metastasizing to the jaw bone [Pathologica 92 (2000) 273].

Intraoperative real-time genetic diagnosis for sentinel node navigation surgery.

Sentinel node navigation surgery (SNNS) has received considerable attention for its role in deciding whether to perform neck dissection in patients with early oral cancer. However, diagnostic accuracy and its intraoperative availability of results remain important concerns. First, we shortened the examination time required for genetic diagnosis. Second, we assessed the quality of the extracted mRNA. Third, 10 patients with early N0 oral cancer underwent SNNS, using our new technique for genetic diagnosis to determine whether neck dissection was required. The examination time of our one-step reverse-transcriptase polymerase chain reaction method using a minicolumn and LightCycler was successfully shortened to 2 h, permitting intraoperative genetic diagnosis. The extracted mRNA was of high quality. Six sentinel nodes in four patients were diagnosed to be metastatic on genetic diagnosis; these patients underwent neck dissection. The other six patients avoided unnecessary surgery. We conclude that intraoperative genetic diagnosis of micrometastasis holds promise of being a sensitive method that can be used to support SNNS.

Transcriptional activation of cyclin-dependent kinase inhibitor, p21waf1 gene by treatment with a differentiation inducing agent, vesnarinone in a human salivary gland cancer cell line.

Recently, a new concept for cancer therapy termed "tumor dormancy therapy" has been proposed. The concept of this therapy is to prolong the survival time of cancer patients while maintaining their quality of life. We have been developing a differentiation-inducing therapy, which is included in the tumor dormancy therapy, for salivary gland cancer. In this study, we examined the effect of a differentiation-inducing drug, Vesnarinone on the growth of several cancer cells, and examined the molecular mechanism by which Vesnarinone induces the cyclin dependent kinase inhibitor, p21waf1 in the cancer cells. Vesnarinone significantly suppressed the growth of TYS (salivary gland cancer cells), PC3 (prostate cancer cells), and A431 (squamous cell cancer cells). Furthermore, Vesnarinone dose-dependently enhanced the expression of p21waf1 mRNA in TYS cells. Using the luciferase reporter assay it was found that the enhancement of p21waf1 mRNA expression by Vesnarinone was through direct transcriptional activation of the p21waf1 promoter. Thus, analyzing the molecular mechanisms of differentiation inducing drugs may lead to the development of a new therapeutic strategy for several human malignancies, including salivary gland cancer.

Vesnarinone, a differentiation inducing drug, directly activates p21(waf1) gene promoter via Sp1 sites in a human salivary gland cancer cell line.

We previously demonstrated that a differentiation inducing drug, vesnarinone induced the growth arrest and p21(waf1) gene expression in a human salivary gland cancer cell line, TYS. In the present study, we investigated the mechanism of the induction of p21(waf1) gene by vesnarinone in TYS cells. We constructed several reporter plasmids containing the p21(waf1) promoter, and attempted to identify vesnarinone-responsive elements in the p21(waf1) promoter. By the luciferase reporter assay, we identified the minimal vesnarinone-responsive element in the p21(waf1) promoter at -124 to -61 relative to the transcription start site. Moreover, we demonstrated by electrophoretic mobility shift assay that Sp1 and Sp3 transcription factors bound to the vesnarinone-responsive element. Furthermore, we found that vesnarinone induced the histone hyperacetylation in TYS cells. These results suggest that vesnarinone directly activates p21(waf1) promoter via the activation of Sp1 and Sp3 transcription factors and the histone hyperacetylation in TYS cells.

Dihydropyrimidine dehydrogenase mRNA level correlates with the response to 5-fluorouracil-based chemo-immuno-radiation therapy in human oral squamous cell cancer.

The measurement of the intra-tumoral level of thymidylate synthetase (TS), and dihydropyrimidine dehydrogenase (DPD), may be useful in predicting tumor sensitivity to 5-fluorouracil (5-FU). In this study, we examined the mRNA levels of DPD and TS in 28 oral squamous cell carcinomas (SCC), and 22 salivary gland tumors by semi-quantitative reverse transcription polymerase chain reaction. Then we examined the correlation of the responsiveness of the patients with oral SCC to 5-FU with the intra-tumoral levels of DPD and TS mRNA. All specimens were obtained at the biopsy before treatment, and then the patients were treated by oral administration of a 5-FU compound (UFT), the irradiation of cobalt-60 (upto 60 Gy) and injection of an immuno-potentiator (OK-432). Intra-tumoral levels of DPD mRNA in the patients who showed CR (complete response) and PR (partial response) were significantly lower than those in the patients who showed NC (no change). However, intra-tumoral levels of DPD mRNA did not correlate with the local recurrence of the tumor during the observation period after initial treatment with or without surgical resection of the residual tumors. On the other hand, TS mRNA levels in the tumors did not correlate with any clinico-pathological parameters. These observations suggest that intra-tumoral levels of DPD mRNA may predict the tumor response to 5-FU-based chemo-immuno-radiation therapy in the patients with oral SCC.

Role of peroxisome proliferator-activated receptor gamma and its ligands in non-neoplastic and neoplastic human urothelial cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors and is expressed in several types of tissue. Although PPARgamma reportedly is expressed in normal urothelium, its function is unknown. We examined the expression of PPARgamma in normal urothelium and bladder cancer in an attempt to assess its functional role. Immunohistochemical staining revealed normal urothelium to express PPARgamma uniformly. All low-grade carcinomas were positive either diffusely or focally, whereas staining was primarily focal or absent in high-grade carcinomas. A nonneoplastic urothelial cell line (1T-1), a low-grade (RT4) carcinoma cell line, and two high-grade (T24 and 253J) carcinoma cell lines in culture expressed PPARgamma mRNA and protein. Luciferase assay indicated that PPARgamma was functional. PPARgamma ligands (15-deoxy-Delta(12,14)-prostaglandin J(2), troglitazone and pioglitazone) suppressed the growth of nonneoplastic and neoplastic urothelial cells in a dose-dependent manner. However, neoplastic cells were more resistant than were nonneoplastic cells. Failure to express PPARgamma or ineffective transcriptional activity may be some of the mechanisms responsible for resistance to the inhibitory action of PPARgamma ligands.

Low-dose retinoic acid enhances in vitro invasiveness of human oral squamous-cell-carcinoma cell lines.

Retinoids inhibit the proliferation of several types of tumour cells, and are used for patients with several malignant tumours. In this study, we examined the effect of retinoic acids (RAs) on the invasive potentials of the oral squamous cell carcinoma (SCC) cells, BHY and HNt. BHY cells expressed all of retinoid nuclear receptors (RARalpha, beta, gamma, and RXRalpha) and cytoplasmic retinoic acid binding proteins (CRABP1 and CRABP2). HNt cells lacked the expression of RARbeta, but expressed other nuclear receptors and CRABPs. All-trans retinoic acid (ATRA) and 13-cis retinoic acid (13-cisRA) (10(-6)and 10(-7)M) inhibited the growth of the cells, but low-dose ATRA and 13-cisRA (10(-8)M) marginally affected the growth of the cells. Surprisingly, low-dose RAs enhanced the activity of tissue-type plasminogen activator (tPA), and activated pro-matrix metalloproteinases (proMMP2 and proMMP9). Activation of proMMP2 and proMMP9 was inhibited by aprotinin, a serine-proteinase, tPA inhibitor. Furthermore, low-dose RAs enhanced the in vitro invasiveness of BHY cells. These results indicate that low-dose RAs enhances the in vitro invasiveness of oral SCC cells via an activation of proMMP2 and proMMP9 probably mediated by the induction of tPA.

Hepatocyte growth factor secreted by prostate-derived stromal cells stimulates growth of androgen-independent human prostatic carcinoma cells.

The objective of the present study is to examine the role of prostate stromal cells on growth and progression of prostate cancer. Co-inoculation of androgen-independent carcinoma cells (PC-3 and CA-7T2) with prostate-derived stromal (P-ST) cells significantly enhanced the growth of carcinoma cells in athymic nude mice. For the in vitro study, a three-dimensional co-culture system was used. It consisted of two layers of collagen gel. Stromal cells were suspended in the lower layer, whereas cancer cells were suspended in the upper layer. Compared to the control culture, the presence of P-ST cells in the lower collagen layer significantly stimulated the growth of carcinoma cells. Such an effect was not demonstrated when carcinoma cells were co-cultured with either bone marrow-derived or skin-derived stromal cells. We identified hepatocyte growth factor (HGF) as the principal growth factor released by P-ST cells but not by bone marrow-derived or skin-derived stromal cells. Neutralizing antibodies against HGF completely abrogated the stimulatory effect of P-ST cells. Exogenous HGF likewise stimulated the growth of carcinoma cells in vitro and in vivo. These results suggest that HGF produced by P-ST cells is a paracrine growth factor that stimulates the growth of androgen-independent prostate cancer cell lines.

Neoplastic conversion of human urothelial cells in vitro by overexpression of H2O2-generating peroxisomal fatty acyl CoA oxidase.

An in vitro study was conducted to determine if malignant transformation can be induced in human urothelial cells immortalized with human papillomavirus E6/E7 genes. A clone designated 1T1 was isolated and then stably transfected with an acyl CoA oxidase (ACOX)-expression construct. The cells generated H2O2 in a large quantity from the substrate linoleic acid (LA). After 56 days of LA treatment, cells persistently formed an epithelial cyst in athymic nude mice with an occasional intracystic epithelial nodule. Our results indicate that human urothelial cells can be transformed to low grade neoplastic cells by H2O2 and suggest that H2O2 may be involved in the development of bladder cancer.

Haematogenous cytokeratin 20 mRNA as a predictive marker for recurrence in oral cancer patients.

We examined the expression of cytokeratin 20 (CK20) mRNA in the peripheral blood of oral squamous cell carcinoma (SCC) patients by reverse transcriptase polymerase chain reaction (RT-PCR). Eleven out of 12 oral SCC patients showed positive RT-PCR results. However, there is no clear relationship between the haematogenous CK20 mRNA and the metastasis. After initial treatment, all of the tumour-free survivors tested showed negative RT-PCR results. CK20 mRNA in peripheral blood can be used as a marker for tumour recurrence but not not for metastasis in oral SCC patients.

Active-MMP2 in cancer cell nests of oral cancer patients: correlation with lymph node metastasis.

We examined the gelatinolytic activity in human oral squamous-cell carcinoma tissues in order to evaluate the capability of intravasation and extravasation of cancer cells. By a microdissection-zymography, we demonstrated separately the gelatinolytic activities in cancer cell nests and stroma adjacent to the cancer cells. The gelatinolytic activities, such as pro-matrix metalloproteinase (MMP)9 and active-MMP2 in most of cancer cell nests were much higher than those of normal gingival epithelium. Moreover, the activities of active-MMP2 in cancer cell nests of metastatic cancers were significantly higher than those of non-metastatic cancers (p<0.05). These results suggest that active-MMP2 in cancer cells can be a predictive marker for metastasis formation in oral squamous-cell carcinoma patients.

Down-regulation of TSC-22 (transforming growth factor beta-stimulated clone 22) markedly enhances the growth of a human salivary gland cancer cell line in vitro and in vivo.

We have recently isolated TSC-22 (transforming growth factor beta-stimulated clone 22) cDNA as a new anticancer drug (Vesnarinone)-inducible gene in a human salivary gland cancer cell line, TYS. We conducted the present study to examine whether up-regulation or down-regulation of TSC-22 can affect the growth of TYS cells in vitro and in vivo. We constructed an expression vector containing sense- or antisense-oriented human TSC-22 cDNA under the transcriptional control of the SR alpha promoter. We cotransfected TYS cells with the sense or antisense expression vector and pSV2neo and obtained more than 200 G418-resistant colonies in each sense or antisense transfectant. Approximately 80% of representative G418-resistant clones expressed the transcripts from transfected sense or antisense TSC-22 cDNA. To avoid the clonal heterogeneity of the cells, we mixed all of the G418-resistant colonies together in each sense or antisense transfectant and examined the expression of TSC-22 protein, in vitro growth, and the tumorigenicity in nude mice. The expression of TSC-22 protein was examined by solid-phase ELISA using a specific antibody against recombinant TSC-22 protein. The expression of TSC-22 protein was up-regulated in the sense transfectants and down-regulated in the antisense transfectants. Contrary to our expectation, up-regulation of TSC-22 protein did not affect both in vitro and in vivo growth of TYS cells. However, down-regulation of TSC-22 markedly enhanced the growth of TYS cells in vitro and in vivo. Furthermore, we examined the expression of TSC-22 mRNA in several human salivary gland tumors. The mRNA expression of TSC-22 in benign and malignant salivary gland tumors was significantly decreased when compared to that in tumor-free salivary glands (P < 0.05; one-way ANOVA), and in some salivary gland tumors, the expression of TSC-22 mRNA was not detectable by reverse transcription-PCR. These results suggest that down-regulation of TSC-22 may play a major role on salivary gland tumorigenesis.

Induction of TSC-22 by treatment with a new anti-cancer drug, vesnarinone, in a human salivary gland cancer cell.

We undertook the present study to clarify the molecular mechanism of the effect of a new anti-cancer drug, vesnarinone, on a human salivary gland cancer cell line, TYS. We isolated TSC-22cDNA as avesnarinone-inducible gene from a cDNA library constructed from vesnarinone-treated TYS cells. TSC-22 was originally reported as a transforming growth factor (TGF)-beta-inducible gene. The expression of TSC-22 was up-regulated within a few hours after treatment with vesnarinone and was continued for 3 days. The level of TSC-22 mRNA in TYS cells was continuously increased until the cells reached confluency. Furthermore, the induction of TSC-22 by vesnarinone was inhibited by treatment with cycloheximide. When we treated the cells with an antisense oligonucleotide against TSC-22 mRNA under quiescent conditions, the antisense oligonucleotide stimulated the growth of TYS cells; however, under growing conditions the antisense oligonucleotide did not affect cell growth. Furthermore, the antisense oligonucleotide suppressed the antiproliferative effect of vesnarinone. These results suggest that TSC-22 may be a negative growth regulator and may play an important role in the antiproliferative effect of vesnarinone.

Possible contribution of active MMP2 to lymph-node metastasis and secreted cathepsin L to bone invasion of newly established human oral-squamous-cancer cell lines.

We have established human oral-squamous-cancer cell lines, BHY and HN, derived from non-metastatic cancer and metastatic cancer respectively. We examined the expression of matrix-degrading enzymes and their inhibitors in these cell lines. Both cell lines expressed pro-matrix metalloproteinase (MMP)1, proMMP2, proMMP9, membrane-type MMP and urokinase-type plasminogen activator. In addition to these enzymes, BHY cells secreted proMMP7 and procathepsin L, while HN cells secreted a large amount of active MMP2. BHY cells secreted a tissue inhibitor of matrix metalloproteinase, TIMP2, but only a trace level of TIMP1. Contrary to BHY cells, HN cells secreted TIMP1, but only a trace level of TIMP2. When we inoculated these cells into the masseter muscle of nude mice, both types of cell formed solid tumors, whose microscopic appearance was identical to that of the original tumors. BHY tumors were highly differentiated squamous-cell carcinomas, and invasive to the masseter muscle and the mandibular bone. Despite their local aggressiveness, BHY tumors did not metastasize to any distant organs. HN tumors were poorly differentiated squamous-cell carcinomas, weakly invasive to the muscle, but not to the mandibular bone. However, HN tumors frequently metastasized to cervical lymph nodes. These results suggest that the net activity of MMP2 (active MMP2/TIMP2) and cathepsin L secreted from cancer cells may contribute respectively to lymph-node metastasis and to bone invasion by oral cancer cells.