Non-curative treatment of patients with oral tongue squamous-cell carcinoma.

PURPOSE: Late-stage OTSCC is associated with poor overall survival (OS). Non-curative treatment approach aims to improve quality of life and prolong survival of patients deemed incurable. The purpose of this study was to investigate the used non-curative treatment modalities for OTSSC and patient survival. METHODS: All patients diagnosed with OTSCC and treated with non-curative intent at the HUS Helsinki University Hospital (Helsinki, Finland) during the 12-year period of 2005-2016 were included. Survival analysis after the non-curative treatment decision was conducted using the Kaplan-Meier method in this population-based study. RESULTS: Eighty-two patients were identified. A non-curative treatment decision was made at presentation without any previous treatment in 26 patients (7% of all patients diagnosed with OTSCC during the study period). Palliative radiotherapy was administered to 24% of all patients. The average survival time after the non-curative treatment decision was 3.7 months (median 2 and range 0-26). CONCLUSIONS: Due to the short mean survival time after decision for treatment with non-curative intent, and the notable symptom burden in this patient population, a prompt initiation of all non-curative measures is warranted.

DIAPH2 alterations increase cellular motility and may contribute to the metastatic potential of laryngeal squamous cell carcinoma.

Low 5-year survival rate in laryngeal squamous cell carcinoma (LSCC) is to large extent attributable to high rate of recurrences and metastases. Despite the importance of the latter process, its complex genetic background remains not fully understood. Recently, we identified two metastasis-related candidate genes, DIAPH2 and DIAPH3 to be frequently targeted by hemizygous/homozygous deletions, respectively, in LSCC cell lines. They physiologically regulate such processes as cell movement and adhesion, hence we found it as a rationale, to study if tumor LSCC specimens harbor mutations of these genes and whether the mutations are associated with metastasizing tumors. As a proof of concept, we sequenced both genes in five LSCC cell lines derived from lymph node metastases assuming there the highest probability of finding alterations. Indeed, we identified one hemizygous deletion (c.3116_3240del125) in DIAPH2 targeting the FH2 domain. Moreover, we analyzed 95 LSCC tumors (53 N0 and 42 N+) using the Illumina platform and identified three heterozygous single nucleotide variants in DIAPH2 targeting conserved domains exclusively in N+ tumors. By combining these results with cBioPortal data we showed significant enrichment of DIAPH2 mutations (P = 0.036) in N+ tumors. To demonstrate the consequences of DIAPH2 inactivation, CRISPR/Cas9 editing was used to obtain a heterozygous DIAPH2+/- mutant HEK-293T cell line. Importantly, the edited line shows a shift from 'proliferation' to 'migration' phenotype typically observed in metastasizing cells. In conclusion, we report that DIAPH2 alterations are present primarily in metastasizing specimens of LSCC and suggest that they may contribute to the metastatic potential of the tumor.

Recurrent CDK1 overexpression in laryngeal squamous cell carcinoma.

In this study, we analyzed the expression profile of four genes (CCNA2, CCNB1, CCNB2, and CDK1) in laryngeal squamous cell carcinoma (LSCC) cell lines and tumor samples. With the application of microarray platform, we have shown the overexpression of these genes in all analyzed LSCC samples in comparison to non-cancer controls from head and neck region. We have selected CDK1 for further analysis, due to its leading role in cell cycle regulation. It is a member of the Ser/Thr protein kinase family of proven oncogenic properties. The results obtained for CDK1 were further confirmed with the application of reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique, Western blot, and immunohistochemistry (IHC). The observed upregulation of CDK1 in laryngeal squamous cell carcinoma has encouraged us to analyze for genetic mechanisms that can be responsible this phenomenon. Therefore, with the application of array-CGH, sequencing analysis and two methods for epigenetic regulation analysis (DNA methylation and miRNA expression), we tried to identify such potential mechanisms. Our attempts to identify the molecular mechanisms responsible for observed changes failed as we did not observe significant alterations neither in the DNA sequence nor in the gene copy number that could underline CDK1 upregulation. Similarly, the pyrosequencing and miRNA expression analyses did not reveal any differences in methylation level and miRNA expression, respectively; thus, these mechanisms probably do not contribute to elevation of CDK1 expression in LSCC. However, our results suggest that alteration of CDK1 expression on both mRNA and protein level probably appears on the very early step of carcinogenesis.

A simple novel prognostic model for early stage oral tongue cancer.

The prognostication of patient outcome is one of the greatest challenges in the management of early stage oral tongue squamous cell carcinoma (OTSCC). This study introduces a simple histopathological model for the prognostication of survival in patients with early OTSCC. A total of 311 cases (from Finland and Brazil) with clinically evaluated early stage OTSCC (cT1-T2cN0cM0) were included in this multicentre retrospective study. Tumour budding (B) and depth of invasion (D) were scored on haematoxylin-eosin-stained cancer slides. The cut-off point for tumour budding was set at 5 buds (low <5; high ≥5) and for depth of invasion at 4mm (low <4mm; high ≥4mm). The scores of B and D were combined into one model: the BD predictive model. On multivariate analysis, a high risk score (BD score 2) correlated significantly with loco-regional recurrence (P=0.033) and death due to OTSCC (P<0.001) in early stage OTSCC. The new BD model is a promising prognostic tool to identify those patients with aggressive cases of early stage OTSCC who might benefit from multimodality treatment.

Matrix metalloproteinase-7 activates heparin-binding epidermal growth factor-like growth factor in cutaneous squamous cell carcinoma.

BACKGROUND: Tumour-specific expression of matrix metalloproteinase (MMP)-7 has been noted in cutaneous squamous cell carcinomas (SCCs) in patients with recessive dystrophic epidermolysis bullosa (RDEB). OBJECTIVES: To examine the potential role of MMP-7 in shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in RDEB-associated and sporadic SCCs. METHODS: Tissue microarrays of RDEB-associated SCC (n = 20), non-EB SCC (n = 60) and Bowen disease (n = 28) were immunostained for MMP-7, CD44 variant 3 (CD44v3) and HB-EGF. Shedding of HB-EGF was studied in vitro using two cutaneous SCC cell lines. RESULTS: Immunohistochemical analysis showed that HB-EGF was absent in tumour cells when MMP-7 and CD44v3 colocalized, and that the absence of HB-EGF was more pronounced in RDEB-associated SCCs than in non-EB SCCs. The loss of HB-EGF in MMP-7-CD44v3 double-positive areas was interpreted to indicate shedding and activation of HB-EGF; this was also detected in Bowen disease indicating its importance in the early phase of SCC development. Specific knockdown of MMP-7 expression in human cutaneous SCC cells by small interfering RNA inhibited shedding of HB-EGF and resulted in diminished activation of the EGF receptor (EGFR) and ERK1/2, and in reduced proliferation of SCC cells. CONCLUSIONS: These findings provide evidence for the role of MMP-7 in promoting the growth of cutaneous SCCs by shedding HB-EGF, and identify EGFR signalling as a potential therapeutic target in RDEB-associated SCC and unresectable sporadic cutaneous SCC.

Morphometric analysis of CD34-positive vessels in salivary gland adenoid cystic and mucoepidermoid carcinomas.

BACKGROUND: Carcinomas of the salivary glands are uncommon and morphologically a diverse group of malignancies. To evaluate the prognostic value of CD34 immunostaining of the vessels in adenoid cystic carcinoma (AdCC) and mucoepidermoid carcinoma (MEC), an automated image analysis method was used. METHOD: In a nationwide study, covering salivary gland cancer (SGC) patients in Finland 1991-1996, 37 AdCC and 18 MEC patients (M 25, F 30, age 25-90, mean 63) were included. In addition to clinical characteristics the size, shape, staining intensity and vessel density in CD34 immunostained histologic samples were measured. RESULTS: Altogether 4433 vessels were measured from AdCC and 2615 from MEC tumor. Of the total tumor vessels measured, 2651 were from patients who deceased with disease (Group I) and 4397 were from specimens derived from those who did not die of disease (Group II) during the 10-year follow-up. The staining intensity was significantly higher in MEC than in AdCC tumor (P = 0.0005). In MEC, the Group I patients had a higher staining intensity among high-grade patients compared with patients with low grade disease, whereas the tumors in Group II had a lower staining intensity among the high-grade compared with the low grade tumors (P = 0.018). A higher vessel density was found in patients with MEC in group II compared with group I (P = 0.017). CONCLUSIONS: The staining intensity of CD34 positive vessels in MEC was higher than in AdCC. In MEC, higher staining intensity of vessels in high-grade tumors and lower vessel density in all MEC patients, predicted poor survival.

Lack of B-RAF mutations in head and neck squamous cell carcinoma.

B-RAF is one of the most commonly mutated oncogenes in human cancer. However, the mutation status of B-RAF has not been established completely in HNSCC. We have analysed the mutation status of the kinase domain of the B-RAF gene (exons 11 and 15) in 91 Japanese HNSCC patients as well as 12 HNSCC cell lines. DNA was extracted and amplified by PCR. Mutations were then analysed by SSCP mutation detection method. Since V600EB-RAF constitutes 90 % of the mutations identified in B-RAF in human cancers, we also used MASA analysis to specifically detect this mutation in exon 15 of B-RAF. Using both methods, no mutation was found in both exon 11 and 15 in all patients and cell lines. Mu tations are absent or rare in the kinase domain of B-RAF in Japanese HNSCC. However, more studies are still needed to determine its usefulness as a target for molecular therapy in these patients.

p38alpha and p38delta mitogen-activated protein kinase isoforms regulate invasion and growth of head and neck squamous carcinoma cells.

Recent studies indicate that the specificity of p38 mitogen-activated protein kinase (MAPK)-mediated cellular stress responses is determined by the expression pattern of the distinct p38 isoforms. Here, we have analysed the function of distinct p38 isoforms in the growth and invasion of head and neck squamous cell carcinomas (HNSCCs). Activation of p38 MAPK by arsenite resulted in inactivation of the ERK1,2 signaling pathway by dephosphorylation of MEK1,2 in primary human epidermal keratinocytes (HEKs), whereas in HNSCC cells this p38-mediated inhibition of the ERK1,2 pathway was absent. Quantitation of p38 pathway component mRNA expression in HNSCC cell lines (n=42) compared to HEKs (n=8) revealed that p38alpha and p38delta isoforms are predominantly expressed in both cell types and that MKK3 is the primary upstream activator expressed. Inhibition of endogenous p38alpha or p38delta activity by adenoviral delivery of corresponding dominant-negative p38 isoforms potently reduced MMP-13 and MMP-1 expressions, and suppressed the invasion of HNSCC cells through collagen. Dominant-negative p38alpha and p38delta inhibited squamous cell carcinoma (SCC) cell proliferation and inhibition of p38alpha activity also compromised survival of SCC cells. p38alpha and p38delta were predominantly expressed in HNSCCs (n=24) and nonneoplastic epithelium in vivo (n=6), with MKK3 being the primary upstream activator. Activation and expression of p38alpha and p38delta by tumor cells was detected in HNSCCs in vivo (n=16). Adenoviral expression of dominant-negative p38alpha or p38delta in cutaneous SCC cells potently inhibited their implantation in skin of severe combined immunodeficiency mice and growth of xenografts in vivo. Our results indicate that p38alpha and p38delta specifically promote the malignant phenotype of SCC cells by regulating cell survival, proliferation and invasion, suggesting these p38 MAPK isoforms as potential therapeutic targets in HNSCCs.

Identification of target genes in laryngeal squamous cell carcinoma by high-resolution copy number and gene expression microarray analyses.

Molecular mechanisms contributing to initiation and progression of head and neck squamous cell carcinoma are still poorly known. Numerous genetic alterations have been described, but molecular consequences of such alterations in most cases remain unclear. Here, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 20 laryngeal cancer cell lines and primary tumors. Our aim was to identify genetic alterations that play a key role in disease pathogenesis and pinpoint genes whose expression is directly impacted by these events. Integration of DNA level data from array-based comparative genomic hybridization with RNA level information from oligonucleotide microarrays was achieved with custom-developed bioinformatic methods. High-level amplifications had a clear impact on gene expression. Across the genome, overexpression of 739 genes could be attributed to gene amplification events in cell lines, with 325 genes showing the same phenomenon in primary tumors including FADD and PPFIA1 at 11q13. The analysis of gene ontology and pathway distributions further pinpointed genes that may identify potential targets of therapeutic intervention. Our data highlight genes that may be critically important to laryngeal cancer progression and offer potential therapeutic targets.

Effect of treatment of larynx and hypopharynx carcinomas on serum syndecan-1 concentrations.

PURPOSE: Syndecan-1 is a multifunctional transmembrane heparan sulfate proteoglycan present on a variety of cell types that mediates basic fibroblast growth factor (bFGF) and other growth factor binding. High serum syndecan-1 (S-syndecan-1) ectodomain levels have been found to be associated with poor outcome in lung cancer and myeloma, but little is known about the effect of cancer treatment on S-syndecan-1 levels. We studied S-syndecan-1 levels longitudinally in a series of patients diagnosed with locoregional squamous cell larynx or hypopharynx carcinoma (n=44) and who we treated with surgery and/or radiation therapy. METHODS: S-syndecan-1 and S-bFGF levels were measured with ELISA prior to, during, and following primary treatment of patients. Syndecan-1 expression was assessed from formalin-fixed and paraffin-embedded tumour samples using immunohistochemistry. RESULTS: S-syndecan-1 levels tended to correlate positively with S-bFGF levels, and the pretreatment levels decreased from a median value of 75 to 58 ng/ml 3 months following treatment (P<0.0001). Patients treated with radiation therapy had a transient increase in S-syndecan-1 during the course of radiation therapy. Patients whose S-syndecan-1 decreased >or=10% from the pretreatment level had more favourable survival than those whose levels remained stable or increased (P=0.0069). Recurred cancer was associated with elevated S-syndecan-1 as compared to the levels measured 3 months following completion of primary therapy. CONCLUSIONS: These findings suggest that a part of S-syndecan-1 originates from the cancerous tissue, and that S-syndecan-1 levels generally decrease following successful cancer treatment.

Activation of Smad signaling enhances collagenase-3 (MMP-13) expression and invasion of head and neck squamous carcinoma cells.

Squamous cell carcinoma (SCC) cells of the head and neck specifically express collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the expression of which correlates with their invasion capacity. Transforming growth factor-beta (TGF-beta) enhances MMP-13 and collagenase-1 (MMP-1) expression and invasion of SCC cells via p38 mitogen-activated protein kinase. Here, we have examined the role of Smad signaling in regulating MMP-13 expression and in invasion of head and neck SCC cells. Treatment with TGF-beta resulted in activation of Smad2 and Smad3 in SCC cells, but had no effect on their proliferation or viability. Basal activation of Smad3 and p38 was noted in SCC cells without exogenous TGF-beta stimulation, and adenoviral delivery of Smad7 and dominant-negative Smad3 inhibited p38 activation in these cells. Adenoviral overexpression of Smad3 augmented the upregulatory effect of TGF-beta on MMP-13 expression by SCC cells. Disruption of Smad signaling by adenoviral expression of kinase-defective TGF-beta type I receptor (activin-receptor-like kinase-5), Smad7, and dominant-negative Smad3 potently suppressed the basal and TGF-beta-induced expression of MMP-13 and MMP-1 in SCC cells, and inhibited their basal and TGF-beta-induced invasion through Matrigel and type I collagen. Adenoviral overexpression of Smad7 in cutaneous and oral SCC cells significantly inhibited their implantation in skin of SCID mice and growth of xenografts in vivo, as compared to LacZ adenovirus-transduced control cells. Together, these results show that Smad signaling plays an important role in promoting the invasive phenotype of human head and neck SCC cells by upregulating their collagenase expression.

Permanent in vitro growth is associated with poor prognosis in head and neck cancer.

OBJECTIVE: The propensity of head and neck carcinomas to grow in vitro and to form a permanent cell line varies. It is not known whether the outcome of patients whose cancer gives rise to permanent in vitro growth differs from that of patients whose cancer cells fail to grow in vitro. The purpose of this study was to find out whether tumor cell capability for in vitro growth is associated with prognosis in head and neck cancer. MATERIAL AND METHODS: The study group consisted of 30 patients treated for head and neck cancer at the University Central Hospital of Turku between 1987 and 1994, and whose tumor samples had produced a permanent cell line in our laboratory. A control group was selected from patients treated during the same time period and with the same protocols in the same department. The controls were selected on the basis of similar tumor localization, TNM status, histological grade, age, gender and general condition. Tumor samples from 14 of the 30 control patients were also cultured, but did not result in a permanent cell line. The median follow-up time was 54 months in the study group and 52 months in the control group. RESULTS: The 3-year survival rate of the patients whose cancer gave rise to in vitro growth was only 19%, compared to 68% among the controls (p = 0.001). In a multivariate analysis the propensity of cancer cells to grow in vitro had independent prognostic value, the relative risk of death (RR) being 1.95 (95% CI 1.11-3.42) when compared to cancers that did not produce a cell line. Of the other factors tested, only the primary tumor size (RR 1.75; 95% CI 0.97-3.16) and the blood hemoglobin level at diagnosis (RR 0.97; 95% CI 0.95-1.01) were possibly independently associated with survival. CONCLUSIONS: The results suggest that the capability of cancer cells for in vitro growth has prognostic significance in head and neck cancer, and that cancer cells that are able to survive and grow in in vitro conditions behave aggressively in vivo. The independence of cancer cells from the paracrine signals produced by the neighboring host cells may enhance cancer cell survival and the metastatic potential in vivo.

Differentiation status of human squamous cell carcinoma xenografts does not appear to correlate with the repopulation capacity of clonogenic tumour cells during fractionated irradiation.

PURPOSE: To investigate the magnitude and kinetics of repopulation in a moderately well differentiated UT-SCC-14 human squamous cell carcinoma [hSCC] in nude mice. This question is of interest because clinical data indicate a higher repopulation capacity in those SCC that have preserved characteristics of differentiation, which appears to be in contrast to results on FaDu and GL hSCC previously reported from this laboratory. METHODS AND MATERIALS: UT-SCC-14 tumours were transplanted subcutaneously into the right hind leg of NMRI nu/nu mice. Fractionated radiation treatments were delivered, either under clamped hypoxia at 5.4 Gy/fraction or under ambient conditions (consistent with an OER of 2.7). Tumours were irradiated every day, every 2nd day, or every 3rd day with 6, 12 or 18 fractions. 1, 2 or 3 days after the last fraction, graded top-up-doses under clamped conditions were given for the purpose of estimating the 50% tumour control dose (TCD50). A total of 22 TCD50 assays were performed and analysed using maximum likelihood techniques. RESULTS: The data demonstrate a slow but significant repopulation of clonogenic cells during fractionated irradiation of UT-SCC-14 hSCC. The results under hypoxic conditions are consistent with a constant repopulation rate, with a clonogenic doubling time (Tclon) of 15.6 days (95% CI: 9.7, 21.4). This contrasts with ambient conditions where Tclon was 68.5 days (95% CI: 124, 161). Both Tclon values are longer than the 6-day volume doubling time of untreated tumours. CONCLUSIONS: Less pronounced repopulation for irradiation under ambient compared to clamped hypoxic conditions might be explained by preferential survival of hypoxic and therefore non-proliferating clonogenic cells. Taken together with previous studies on poorly differentiated FaDu and moderately well differentiated GL hSCC, the results are consistent with considerable variability in the magnitude and kinetics of repopulation in different experimental squamous cell carcinomas, and with a relationship between reoxygenation and repopulation during fractionated irradiation. The differentiation status of hSCC growing in nude mice does not to appear to correlate with the proliferative capacity of clonogenic tumour cells during treatment. The results do not support the hypothesis gained from clinical data of higher repopulation in well-differentiated tumours.

Expression and regulation of collagenase-2 (MMP-8) in head and neck squamous cell carcinomas.

MMP-8 (collagenase-2) is the most effective collagenase to initiate type I collagen degradation. Since initiation of lysis of the surrounding collagen matrix is an essential prerequisite for carcinoma cells to spread, this study investigated the expression of MMP-8 in squamous cell carcinoma (SCC) of the head and neck in vivo and in vitro. Most of the recently established head and neck carcinoma cell lines (22/25), corresponding tumour (5/7) and dermal (2/2) fibroblasts, commercial tongue carcinoma (HSC-3 and SCC-25), and transformed keratinocyte cell lines of the tongue (IHGK) and skin (HaCaT) expressed MMP-8 mRNA analysed by the PCR method. Western blotting revealed a latent 50 kD band in concentrated culture media of carcinoma cells and corresponding tumour and dermal fibroblasts. The expression of immunoreactive MMP-8 protein was reduced 30% by transforming growth factor beta-1 (TGF-beta1) at 1 ng/ml concentration and 60% at 10 ng/ml concentration, but up-regulated 2- and 2.5-fold after 10 nM and 100 nM phorbol 12-myristate 13 acetate (PMA), respectively. Immunohistological staining localized MMP-8 protein in a few malignant invading tumour cell islands, certain fibroblasts, polymorphonuclear neutrophils (PMNs), and plasma cells. In situ hybridization revealed a faint sporadic signal in carcinoma cells of all eight tissue sections analysed. It is concluded that tissue from head and neck carcinomas can express MMP-8 both in vivo and in vitro. Since the amount of MMP-8 in carcinoma and stromal cells is rather low, MMP-8 may have a potential role, with other collagenases, in the proteolysis of connective tissue associated with the spreading of invasive carcinoma.

Expression and regulation of MMP-20 in human tongue carcinoma cells.

Human matrix metalloproteinase-20 (MMP-20, enamelysin) fragments the enamel-specific protein amelogenin and has been shown to be synthesized exclusively by odontoblasts and ameloblasts and in certain odontogenic tumors. Here we demonstrate, for the first time, the expression of MMP-20 mRNA and protein in two carcinoma cell lines originating from the tongue. Treatment of the SCC-25 and HSC-3 cells with phorbol 12-myristate 13-acetate (10 nmol/L) up-regulated MMP-20 mRNA and protein expression by up to 1.6-fold, but transforming growth factor beta (10 ng/mL) had no effect. The latent proform of recombinant (r) human MMP-20 was converted by tumor-related trypsin-2. Activated rMMP-20 did not degrade type I or type II collagen, but efficiently hydrolyzed fibronectin, type IV collagen, laminin-1 and -5, tenascin-C, and beta-casein. This implies that MMP-20 not only participates in dental matrix remodeling but is also present in tongue carcinoma cells.

Imaging of blood flow and hypoxia in head and neck cancer: initial evaluation with [(15)O]H(2)O and [(18)F]fluoroerythronitroimidazole PET.

UNLABELLED: Hypoxia is a characteristic feature of malignant tumors that should be evaluated before the start of therapy. (18)F-labeled fluoroerythronitroimidazole (FETNIM) is a possible candidate for imaging tumor hypoxia with PET. Quantitative analysis of [(18)F]FETNIM uptake in vivo is necessary before proceeding to assays predicting hypoxia. METHODS: Eight patients with untreated head and neck squamous cell carcinoma were enrolled in the study. All patients underwent dynamic PET imaging with [(18)F]FETNIM, coupled with measurements of blood flow with [(15)O]H(2)O and blood volume with [(15)O]CO. The metabolically active tumor volume was determined from [(18)F]FDG PET performed on a separate day. [(18)F]FETNIM uptake in the tumor was correlated with that in neck muscles and arterial plasma and compared with the findings of other PET studies. RESULTS: Blood flow in tumor was 5- to 30-fold greater than in muscle, in contrast to blood volume, which did not significantly differ in the 2 tissues. With [(18)F]FETNIM PET, muscle activity remained invariably less than plasma activity, whereas activity in whole tumors was always greater than that in muscle. In 4 instances, the maximum tumor uptake of [(18)F]FETNIM was 1.2-2.0 times higher than plasma activity in the late dynamic phase. A kinetic model developed for calculation of distribution volume of reversibly trapping tracers was successfully applied in the [(18)F]FETNIM studies. Tumor distribution volume correlated strongly with the standardized uptake value of [(18)F]FETNIM between 60 and 120 min and with blood flow but not with the standardized uptake value of [(18)F]FDG. The relationship between [(18)F]FETNIM uptake and the blood flow of the tumor was less obvious on a pixel-by-pixel level. CONCLUSION: Uptake of [(18)F]FETNIM in head and neck cancer is highly variable and seems to be governed by blood flow at least in the early phase of tissue accumulation. Maximum tumor-to-muscle tracer uptake ratios > 180 min were in the range of 1-4, comparing favorably with those reported previously for [(18)F]fluoromisonidazole. Assessment of the distribution volume of [(18)F]FETNIM after the initial blood-flow phase is feasible for subsequent evaluation of hypoxia-specific retention.

A follow-up study of patients suffering from sudden sensorineural hearing loss.

Sudden sensorineural hearing loss (S-SNHL) is a common problem with a high recovery rate. However, little is known of the long-term prognosis of affected patients. The purpose of this follow-up study was to evaluate the long-term hearing results of S-SNHL patients. The sample consisted of 168 patients with S-SNHL treated with carbogen inhalation and/or anticoagulant therapy during the period 1982-89. A questionnaire was sent to these patients, and audiological investigations were carried out in a selection of these patients in 1997. Comparison of the different treatment methods showed that the difference observed in improvement of hearing was statistically significant between the carbogen inhalation and anticoagulant treatment groups. The hearing improvement achieved was stable for, on average, 8 years of follow-up. During the follow-up period, Ménière's disease was diagnosed in only 1 of the 116 patients who answered the questionnaire and no cases of acoustic neurinoma were diagnosed, indicating that establishment of a careful patient history and clinical and audiological investigations are sufficient for the diagnosis of S-SNHL. In general, the hearing improvement achieved in S-SNHL patients is stable during long-term follow-up.

Mannose receptor is a novel ligand for L-selectin and mediates lymphocyte binding to lymphatic endothelium.

Continuous lymphocyte recirculation between blood and lymphoid tissues forms a basis for the function of the immune system. Lymphocyte entrance from the blood into the tissues has been thoroughly characterized, but mechanisms controlling lymphocyte exit from the lymphoid tissues via efferent lymphatics have remained virtually unknown. In this work we have identified mannose receptor (MR) on human lymphatic endothelium and demonstrate its involvement in binding of lymphocytes to lymphatic vessels. We also show that the binding requires L-selectin, and L-selectin and MR form a receptor-ligand pair. On the other hand, L-selectin binds to peripheral lymph node addressins (PNAds) on high endothelial venules (HEVs) that are sites where lymphocytes enter the lymphatic organs. Interestingly, MR is absent from HEVs and PNAds from lymphatic endothelium. Thus, lymphocyte L-selectin uses distinct ligand molecules to mediate binding at sites of lymphocyte entrance and exit within lymph nodes. Taken together, interaction between L-selectin and MR is the first molecularly defined mechanism mediating lymphocyte binding to lymphatic endothelium.

Core 2 beta1,6-N-acetylglucosaminyltransferases and alpha1,3-fucosyltransferases regulate the synthesis of O-glycans on selectin ligands on oral cavity carcinoma cells.

Selectin-dependent cell binding has importance in the extravasation of blood-circulating tumor cells and in the generation of metastases. Cell surface glycoproteins decorated with sialylated, fucosylated epitopes, such as sialyl Lewis(x) (sLe(x)), are ligands for selectins. Not only terminal sLe(x) moieties but also proximal core structures contribute to the formation of binding epitopes for selectins. Core 2 beta1,6-N-acetylglucosaminyltransferases (C2GnT) and alpha1,3-fucosyltransferases (alpha1,3-FucT) have been suggested to be the rate-limiting enzymes in the synthesis of selectin ligands. We analyzed oral cavity epithelial carcinoma cell lines and showed their expression of RNA transcripts for C2GnT and alpha1,3-FucT, identified alpha1,3-FucT enzyme activities, and analyzed the cell surface sLe(x) expression levels. Neither the pattern of expressed enzymes nor the alpha1,3-FucT activity directly predicted the binding capacity of E-selectin. However, only the sLe(x)-expressing cell lines were capable of binding to E-selectin, but not to P-selectin, thus putatively promoting the selectin-mediated metastasis. These findings suggest that C2GnT in combination with alpha1,3-Fuc-T contribute to the selectin-mediated metastasis in oral cavity carcinomas.

Endoscopic laser surgery for laryngeal cancer.

Endoscopic laser surgery is a novel treatment modality for laryngeal cancer. CO2 laser combined with an operating microscope is the most frequently used instrumentation. In Finland we started large-scale laser surgery in 1994 in all five university hospitals, covering a population of about five million people. By 1998 we had operated on 140 patients, of whom 11 were females. Eighty-three per cent of the lesions were glottic. Because of the low number of stage III-IV patients, the recurrence and survival analyses included 132 patients with in situ, stage I or stage II tumours, numbering 8, 96 and 28 respectively. The mean follow-up time was 38 months. The 2-year recurrence frequencies were 5% for stage I, 31% for stage II, and 11% altogether. No patients developed recurrences after 2 years. Seven patients underwent a salvage laryngectomy and the adjusted cumulative survival rate was 95%. After laser surgery the quality of voice was good or excellent in 70% and only three patients suffered from severe aphonia. This study showed that the results of endoscopic laser surgery are comparable with those of radiation therapy, but this type of treatment is more convenient for the patients and much cheaper for society.