Search

Background: The cellular composition of the tumor microenvironment (TME) at the invading front of oral squamous cell carcinomas (OSCCs) may reflect biologically important cancer features and host responses, and thus be related to disease progression. The TME density of mast cells (MCs), macrophages, cancer-associated fibroblasts (CAFs) and endothelial cells were quantified at the invasive front and analyzed regarding their relation to disease recurrence in patients with small T1/2N0M0 OSCCs. mRNA for MC-specific proteins were analyzed in a second patient cohort with head and neck squamous cell carcinoma (HNSCC). Materials and Methods: Samples from 62 patients with T1/2N0M0 OSCC were immunohistochemically stained and scored for the cellular expression of mast/stem cell growth factor receptor (c-KIT) (MCs), CD68 (macrophages), α-smooth muscle actin (α-SMA) (CAFs) and CD31 (endothelial cells) and this was analyzed according to disease recurrence. Data from The Cancer Genome Atlas database were used to examine mRNA expression profiles and clinical data of patients with 399 HNSCC. Results: Increased MC density at the invasive front was significantly associated with reduced disease recurrence, as none of the patients with high MC density experienced relapse. Moreover, increased expression of mRNA for MC specific markers as c-KIT, and α-, β-, and δ-tryptases and the MC-stimulating factor, stem cell factor (SCF), was significantly associated with good prognosis in patients with HNSCC. Conclusion: Decreased MC density at the invasive front may reflect tumor biology related to disease progression and prognosis. Counting MCs seems to be an easy and practical tool, that could be utilized for prognostic evaluation.

The six microRNAs (miRNA) encoded by the miR-17–92 cluster, also named oncomir-1, have been associated with carcinogenesis and typically exhibit-increased expression in tumors. Despite the well-established role for the miR-17–92 cluster in an oncogenic network, the physiological function of these miRNAs in normal tissues remains unresolved. In order to investigate whether there are similar patterns of miR-17–92 expression during embryogenesis and carcinogenesis, we have preformed a systematic study of the expression in cultured carcinoma cells, cultured primary human keratinocytes (KC), and during development of some murine tissues. Both levels of expression of the primary transcript (pri-miRNA) and levels of expression of the individual members of the cluster were monitored. Irrespectively of tissue examined we found that the level of expression decreased markedly during development. With cultured primary human KCs their levels of expression of some of these microRNAs decreased as the number of cell passages increased. Their levels of expression in cultured carcinoma cells, in contrasts, increased, or remained unchanged, with increasing number of cell passages. The results suggest these microRNAs are involved in the regulation of foetal development and that they may promote proliferation and inhibit differentiation during embryogenesis and carcinogenesis. Additionally, the six microRNAs exhibit variable tissue expression, suggesting selective processing of these microRNAs. J. Cell. Physiol. 226: 2257–2266, 2011. © 2010 Wiley-Liss, Inc.

Brusevold IJ, Soland TM, Khuu C, Christoffersen T, Bryne M. Nuclear and cytoplasmic expression of Met in oral squamous cell carcinoma and in an organotypic oral cancer model. Eur J Oral Sci 2010; 118: 342–349. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci Met, the hepatocyte growth factor receptor, is important in transducing signals for tumour growth and metastasis. The aim of this study was to examine the pattern of Met expression and its value as a prognostic factor in oral squamous cell carcinomas (OSCCs). The material consisted of 53 OSCCs and five healthy controls from normal oral mucosa supplied with cell lines, 10 organotypic models supplied with oral cancer cells, and three organotypic models supplied with normal keratinocytes. Met protein expression was assessed by immunohistochemistry and western blotting. Met expression was scarce and limited to the basal layer in normal oral mucosa, but was more extensive in the tumours. Cytoplasmic expression of Met was found in the majority of the tumours, and nuclear expression was found in 72%, including a high fraction of the cells located at the invasive front. Organotypic models with normal or malignant oral cells yielded principally similar results as in the mucosa and the cancers, respectively. A smaller amount of Met immunoreactivity was detected, by western blotting, in the nuclear fraction of cultured oral cancer cells. In conclusion, Met was upregulated in OSCCs and was also found in the nucleus. However, Met was not a marker for prognosis in this study.

The intracellular signalling cascade(s) mediating epidermal growth factor (EGF)-induced cyclooxygenase-2 (COX-2) expression is poorly defined in oral carcinomas. Investigation of two different oral squamous cell carcinoma (OSCC) cell lines with high EGF-induced COX-2 expression revealed, however, that this expression was dependent on two mitogen-activated protein kinase (MAPK) pathways [extracellular signal-regulated kinase 1/2 (ERK1/2) and p38] because combined inhibition of these pathways was needed to abolish EGF-induced COX-2 expression. Surprisingly, inhibition of phosphoinositide-3 kinase (PI3K) increased EGF-induced COX-2 expression in the basaloid OSCC cell line (C12), suggesting a PI3K-controlled, inhibitory COX-2-regulating pathway. Neither the transcription factor nuclear factor-kappaB (NF-kappaB), nor Src, was involved in EGF-induced COX-2 expression. The results suggest that EGF-induced COX-2 expression is regulated by several pathways, and emphasizes that individual tumors use different strategies for intracellular signalling.

Aims To evaluate the expression of p75 neurotrophin receptor (p75(NTR)) in oral squamous cell carcinoma (OSCC). The results were related to tumour node metastasis (TNM) stage, World Health Organization (WHO) grade, invasive front grading (IFG) and prognosis. Methods and results Immunohistochemically, the expression of p75(NTR) was assessed in 53 T1-T2 OSCCs. Clinical data were recorded prospectively. The end-point was disease-free survival. All tumours expressed p75(NTR), and this expression, both in central/superficial tumour areas and at the invasive front, was associated with poor prognosis (P = 0.03 and P = 0.02) (log rank test). Tumours with marked cellular dissociation (IFG parameter) had more recurrences than tumours with collective tumour cell invasion (P = 0.03). In tumours showing both p75(NTR) at the invasive front and marked tumour cell dissociation, the average risk of recurrence was increased about 17 times (Cox regression analysis) compared with tumours with low p75(NTR) expression and collective invasion. Traditional prognostic systems were of no prognostic significance. Conclusion p75(NTR) was expressed in all OSCCs. p75(NTR) expression and the pattern of invasion were significantly associated with a poor prognosis in OSCCs, and both were better prognostic factors than traditional prognostic parameters. The combination of p75(NTR) expression and the pattern of invasion strongly increased precision in the identification of tumours with poor disease-free survival.

Trefoil factor 3 (TFF3) is a member of the mammalian TFF family. Trefoil factors are secreted onto mucosal surfaces of the entire body and exert different effects according to tissue location. Trefoil factors may enhance mucosal healing by modulating motogenic activity, inhibiting apoptosis, and promoting angiogenesis. Trefoil factor 3 is secreted from the submandibular gland and is present in whole saliva. The aim of this study was to assess the migratory and proliferative effects of TFF3 on primary oral human keratinocytes and oral cancer cell lines. The addition of TFF3 increased the migration of both normal oral keratinocytes and the cancer cell line D12, as evaluated by a two-dimensional scratch assay. By contrast, no increase in proliferation or energy metabolism was observed after stimulation with TFF3. Trefoil factor 3-enhanced migration was found to be driven partly by the extracellular signal-related kinase (Erk1/2) pathway, as shown by addition of the mitogen-activated protein kinase (MAPK) inhibitor PD 98059. Previous functional studies on trefoil peptides have all been based on cells from monolayered epithelium like the intestinal mucosa; this is the first report to show that normal and cancerous keratinocytes from stratified epithelium respond to TFF stimuli. Taken together, salivary TFF3 is likely to contribute to oral wound healing.

Background: Histomorphological grading at the invasive front of oral squamous cell carcinomas (OSCCs) may provide useful prognostic information. In the present study, we investigated the presence and prognostic value of activated phosphorylated extracellular signal-regulated kinases 1 and 2 (p-ERK1/2) and cyclo-oxygenase-2 (COX-2) both at the invasive front and in central/superficial parts of OSCCs. Methods: Using immunohistochemistry, we assessed the presence of p-ERK1/2 and COX-2 in 53 early stage OSCCs. Clinical data were recorded prospectively. The end point was disease-free survival. Results: p-ERK1/2 staining was present in almost all tumours. The staining was mostly nuclear in the cells of the invasive front and either nuclear or nuclear/cytoplasmic in central/superficial tumour parts. COX-2 was observed in almost all tumours (98%) and the staining was often restricted to focal areas. Most tumours were COX-2 negative at the invasive front. The lowest P-value in survival analyses was P = 0.06 for p-ERK1/2 at the invasive front. COX-2, the histomorphological grading systems and TNM stage were of no prognostic value. Conclusion: p-ERK1/2 was present in almost all tumours and p-ERK1/2 may be a prognostic marker at the invasive front of OSCCs. In early stage OSCCs, most tumours did not express COX-2 at the invasive front.

Early oral carcinomas have a high recurrence rate despite surgery with clear margins. In an attempt to classify the risk of recurrence of oral squamous cell carcinomas, we explored the significance of tumor budding, epithelial-mesenchymal transition (EMT) and certain cancer stem cell markers (CSC).Tumor budding (single cells or clusters of ≤5 cells in the tumor front, divided into high- and low-budding tumors), EMT and CSC markers were studied in 62 immunohistochemically stained slides of T1/2N0M0 oral squamous cell carcinomas. Tissues and records of follow-up were obtained from the Oslo University Hospital, Norway. Tumor budding, EMT and CSC markers were scored and analyzed.The only significant prognostic marker was tumor budding (p=0.043). Expression of the EMT marker E-cadherin was lost from the invasive front and tended to be a prognostic factor (p=0.17), and up-regulation of vimentin in tumor cells in the invasive front was found; this indicates that EMT had occurred. CSC markers were not associated with recurrence rate in the present study.A high budding index was related to poor prognosis in patients with oral cancer. Budding was associated with EMT-like changes. CSC factors were detected but reflected differentiation rather than stemness. Scoring of buds in patients with oral cancer may help discriminate invasive tumors prone to relapse, and thus, provide an indication for adjuvant therapy.

Background Although the standard treatment of oral leukoplakia ranges from watchful waiting to complete resection, the value of these approaches is unknown. Methods We studied the relations among resection, ploidy status, and death from cancer in 103 patients with diploid dysplastic oral leukoplakia, 20 patients with tetraploid lesions, and 27 patients with aneuploid lesions. Data on cancer-specific mortality and treatment were obtained from the Cancer Registry of Norway, Statistics Norway, and chart reviews. Results Primary oral carcinoma developed in 47 of the 150 patients with leukoplakia (31 percent) — 5 with diploid, 16 with tetraploid, and 26 with aneuploid leukoplakia — during a mean follow-up of 80 months (range, 4 to 237). The margin status of the initial leukoplakia resection had no relation to the development of oral cancer (P=0.95). Twenty-six of the 47 patients in whom cancer developed (4 with prior tetraploid and 22 with prior aneuploid lesions) had recurrences (55 percent); the recurrences ...

This article has been retracted at the request of Oral Oncology and the co-authors. Please see http://www.elsevier.com/locate/withdrawalpolicy . Reason: The lead author, Jon Sudbo, has been found by the Investigating Commission (appointed in January 2006 by Rikshospitalet - Radiumhospitalet MC and the University of Oslo to investigate Dr Sudbo's misconduct) to have fabricated and modified the data on which the analyses were based. The above mentioned Investigating Commission has determined that Dr Sudbo acted alone in fabricating the data. Dr Sudbo disputes the findings of the Investigating Commission. Reference: Ekbom A, Helgesen GEM, Lunde T, Tverdal A, Vollset SE, Simonsen S. Report from the Investigating Commission appointed by Rikshospitalet - Radiumhospitalet MC and the University of Oslo, January 18, 2006.

OBJECTIVES: The role of cyclooxygenase-2 (COX-2) in disease has been extensively studied, (Annu Rev Med (2002) 53:35; N Engl J Med (2001) 345:433) but less information is available with respect to possible physiological functions of COX-2. Information on how and where COX-2 is expressed under physiological conditions may increase our understanding of its physiological role. Previous studies have revealed a COX-2 dependent production of prostanoids under physiological conditions, without entirely determining the source of this production. MATERIALS AND METHODS: To assess COX-2 expression under normal conditions, we analyzed tissue specimens that were removed from 30 healthy study subjects in conjunction with surgical procedure related to insertion of dental implants and from three patients which had muscle tissue from Quadriceps femoris muscle removed as part of surgical treatment of soft tissue sarcomas not directly affecting the muscle tissue. Immunohistochemistry and immunoblotting (Western blotting) was used to assess the presence of COX-2 protein. RESULTS: In 25 of 30 patients (83%), COX-2 protein was expressed in striated muscle, as assessed by immunohistochemistry. All cases had COX-2 expression verified by Western blotting. In none of the 25 subjects with COX-2 expression did we notice concomitant inflammation of the adjacent submucosal tissue. CONCLUSIONS: It is a novel finding that COX-2 is expressed in striated muscle under physiological conditions. COX-2 activity in striated muscle is a possible explanation for the hitherto unknown localization of prostanoids synthesis under physiological conditions.

Emerging data indicate a link between genetic instability and up-regulation of cyclooxygenase-2 (COX-2). To see if individuals at high risk of oral cancer are candidates for treatment with selective COX-2 inhibitors (coxibs), levels of COX-2 expression in healthy, premalignant and cancerous oral mucosa were compared with the occurrence of DNA ploidy status as a genetic risk marker of oral cancer. COX-2 gene product was evaluated immunohistochemically in 30 healthy persons, in 22 patients with dysplastic lesions without previous or concomitant carcinomas, and in 29 patients with oral carcinomas. The immunohistochemical findings were verified by western blotting. COX-2 expression was correlated to DNA content as a genetic risk marker of oral cancer. COX-2 was up-regulated from healthy to premalignant to cancerous oral mucosa. Thus, COX-2 expression was found in 1 case of healthy oral mucosa (3%). All specimens from healthy mucosa had a normal DNA content. In patients with premalignancies. In 29 patients with oral carcinomas, cyclooxygenase-2 expression was observed in 26 (88%), and aneuploidy was observed in 25 cases (94%, P=0.04). Notably, of 22 patients with dysplastic lesions, COX-2 was exclusively expressed in a subgroup of nine patients (41%) identified to be at high risk of cancer by the aberrant DNA content of their lesions. Seven of these patients were followed for 5 years or more. An oral carcinoma developed in six of them (85%; P=0.02). These findings emphasize the need to determine whether coxibs can reduce the risk of oral cancer in patients with high-risk precancerous lesions.

We studied the role of caveolin-1 in tumor progression and prognosis in serous ovarian carcinoma and the association between caveolin-1 and MDR1 expression. The study involved immunohistochemical analysis for caveolin-1 and P-glycoprotein (P-gp) expression in 75 effusions and 90 solid lesions from ovarian and primary peritoneal carcinoma; in situ hybridization for MDR1 messenger RNA (mRNA) expression in 62 effusions and all 90 tumors; and reverse transcription-polymerase chain reaction (RT-PCR) for caveolin-1 mRNA expression in 23 effusions. Immunohistochemical analysis localized caveolin-1 to the cell membrane in 43 effusions and 24 tumors. P-gp membrane expression was detected in 14 effusions and 11 tumors; MDR1 mRNA, in 20 effusions and 30 tumors. Caveolin-1 mRNA was expressed in 19 effusions. Caveolin-1 protein expression showed no association with that of P-gp protein or MDR1 mRNA. The expression of all markers was similar in carcinoma cells in pleural and peritoneal effusions. Caveolin-1 is a novel diagnostic marker for effusions; expression is moderately elevated in tumor cells in effusions, possibly owing to altered signal transduction and metabolism in cancer cells at this site. Expression seems MDR1 independent.

Angiogenic factors are involved in tumor growth and spread. The aim of this study was to evaluate the expression of angiogenesis-related genes in malignant serous effusions of patients with advanced-stage (FIGO stage III and IV) ovarian carcinoma. In addition, to compare the results for carcinoma cells in effusions with corresponding primary tumors and metastatic lesions, and analyze their prognostic role. Sections from 66 effusions and 90 primary and metastatic lesions from 62 ovarian and primary peritoneal carcinoma patients, were evaluated for expression of basic fibroblast factor (bFGF), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) using mRNA in situ hybridization (ISH). Protein expression was evaluated in a subset of specimens using immunohistochemistry (IHC). ISH results were correlated with clinical parameters. In both effusions and solid tumors, bFGF mRNA was the most commonly expressed factor (93% of effusions and 95% of solid tumors) followed by IL-8, while VEGF was expressed in a minority of the specimens (P 0.05). Peritoneal and pleural effusions showed similar expression patterns. In conclusion, bFGF is the major angiogenic factor expressed in ovarian carcinoma at the mRNA level. It is highly expressed in both solid tumors and serous effusions, while IL-8 and VEGF are down regulated in carcinoma cells in effusions, possibly due to the lack of interaction with stromal cells. mRNA expression of VEGF, bFGF, and IL-8 does not appear to be a predictor of disease outcome in advanced-stage ovarian carcinoma. Carcinoma cells in pleural and peritoneal effusions show a similar metastatic expression profile, in agreement with our previous findings, supporting the true metastatic nature of ovarian carcinoma cells in ascites.

Objective Expression of the stem cell transcription factor SOX2 is often used to imply stemness and poor prognosis in cancer. However, its role in oral squamous cell carcinoma (OSCC) is not fully elucidated. Material and methods Tumour tissues from 62 patients with primary, node negative and non-metastatic OSCCs were used to evaluate SOX2 expression by immunohistochemistry. The results were correlated to clinicopathology, treatment and disease recurrences. Results The majority of the OSCCs (88%) expressed SOX2. Patients with higher nuclear SOX2 staining intensity in the invasive front compared to the adjacent normal epithelium, had a remarkable longer disease-free period if they received adjuvant post-operative radiotherapy (P = 0.001). This was in particular evident for highly differentiated OSCCs, as none of the high SOX2-expressing tumours reoccurred in contrast to all low SOX2-expressing OSCCs. Conclusions High nuclear SOX2 expression in the invasive front was associated with dramatic longer disease-free period than low SOX2-expressing carcinomas after post-operative radiotherapy in small OSCCs. The result suggested that high nuclear SOX2 expression at the invasive front may predict radiosensitivity.

Objective. The aim of this study was to investigate the expression of cell cycle proteins in ovarian carcinoma cells in serous effusions and respective solid tumors. Methods. Fifty-five malignant effusions and 38 tumors (20 primary, 18 metastatic) were immunohistochemically stained for cyclin A, p27 kip1 , and Ki-67. Staining extent (0–100% cells) and intensity (0–3 scale) were scored. Cyclin A and p27 kip1 expression was additionally studied in 29 malignant effusions using immunoblotting. Immunohistochemistry results in effusions were evaluated for possible association with clinicopathologic parameters. Results. Nuclear immunoreactivity for all markers was detected on carcinoma cells in the majority of effusions using immunohistochemistry. Similarly, immunoblotting showed the presence of cyclin A and p27 kip1 in 29/29 and 25/29 specimens, respectively. Intense (3) immunoreactivity for Ki-67 was detected more often in peritoneal effusions, compared with those of pleural location ( P = 0.036). Staining in primary and metastatic lesions was generally comparable to that of tumor cells in effusions. Staining for p27 kip1 was more diffuse in effusion specimens obtained prior to the institution of chemotherapy ( P = 0.042). In an analysis of all effusions, an association was observed between the number of cells that were immunoreactive for Ki-67, cyclin A, and p27 kip1 (cyclin A–Ki-67: P = 0.008; p27 kip1 –Ki-67: P = 0.019; cyclin A–p27 kip1 : P = 0.032). In survival analysis, the presence of more diffuse ( P = 0.042) and intense ( P = 0.019) staining for cyclin A correlated with prolonged overall survival. Conclusions. The expression of the studied cell cycle markers does not differ markedly between ovarian carcinoma cells in the pleural and peritoneal cavity, supporting our previous studies of several metastasis-associated molecules. The presence of cyclin-A-positive cell populations is associated with a more favorable disease outcome, possibly due to the targeting of proliferating cells by chemotherapeutic agents. However, the decline in the fraction of p27 kip1 -positive cells in posttreatment specimens may point to additional mechanisms involved in this selection.

This article has been retracted. See retraction notice [DOI: 10.1002/path.2115]. Approximately one in ten oral white patches (leukoplakia) are histologically classified as dysplasia, with a well-documented potential for developing into oral squamous cell carcinoma (OSCC). Histological grading in oral dysplasia has limited prognostic value, whereas large-scale genomic status (DNA ploidy, nuclear DNA content) is an early marker of malignant transformation in several tissues. Biopsies from 196 patients with oral leukoplakia histologically typed as dysplasia were investigated. Inter-observer agreement among four experienced pathologists performing a simplified grading was assessed by Cohen's kappa values. For 150 of the 196 cases, it was also possible to assess large-scale genomic status and compare its prognostic impact with that of histological grading. Disease-free survival was estimated by life-table methods, with a mean follow-up time of 103 months (range 4‒165 months). The primary considered end-point was the subsequent occurrence of OSCC. For grading of the total of 196 cases, kappa values ranged from 0.17 to 0.33 when three grading groups (mild, moderate and severe dysplasia) were considered, and from 0.21 to 0.32 when two groups (low grade and high grade) were considered (p = 0.41) . For the 150 cases in which large-scale genomic status was also assessed, kappa values for the histological grading ranged from 0.21 to 0.33 for three grading groups and from 0.27 to 0.34 for two grading groups (p = 0.47). In survival analysis, histological grading was without significant prognostic value for any of the four observers (p 0.14 ‒ 0.44), in contrast to DNA ploidy (p = 0.001). It is concluded that DNA ploidy in oral dysplasia has a practical prognostic value, unlike histological grading of the same lesions. Copyright © 2001 John Wiley & Sons, Ltd.

Objective. The aim of this study was to analyze the correlation among the expression of caveolin-1, the protein constituent of caveolae, and disease outcome in advanced-stage ovarian carcinomas. Methods. Sections from 76 primary ovarian carcinomas and metastatic lesions from 45 patients diagnosed with advanced-stage ovarian carcinoma (FIGO stages III–IV) were evaluated for caveolin-1 expression using immunohistochemistry. Patients were divided into long-term survivors and short-term survivors based on disease outcome. Twenty nonneoplastic fallopian tubes and ovaries were additionally studied. Results. The mean follow-up period was 70 months. The mean values for disease-free survival and overall survival were 109 and 125 months for long-term survivors, compared to 3 and 21 months for short-term survivors, respectively. Caveolin-1 expression was localized to the cell membrane in 24/76 (32%) specimens and was detected in the cytoplasm in 52/76 (68%) cases. Both patterns were more often detected in metastases, when compared with primary tumors. In addition, membrane immunoreactivity was more often seen in tumor of short-term survivors. These differences did not reach statistical significance ( P > 0.05). Combined membrane and cytoplasmic immunoreactivity was seen in 17/20 (85%) nonneoplastic lesions. Despite its role in tyrosine-kinase-mediated signal transduction in vitro studies, caveolin-1 expression in carcinomas showed no association with the protein expression of c-erbB-2 and epidermal growth factor receptor, evaluated in a previous study of this patient cohort. Conclusions. This study provides the first in vivo evidence of caveolin-1 membrane expression in human malignancies. Caveolin-1 is often expressed in advanced-stage ovarian carcinoma, but does not appear to be a powerful predictor of disease outcome in these tumors. The reduced expression level in carcinomas compared to nonneoplastic epithelium may point to a role for caveolin-1 as a tumor suppressor gene.

Objective. The aim of this study was to analyze the correlation between expression of E-cadherin complex proteins, epidermal growth factor receptor (EGFR), and c-erbB-2 and disease outcome in advanced-stage ovarian carcinomas. Methods. Sections from 75 primary ovarian carcinomas (=37) and metastatic lesions (=38) from 45 patients diagnosed with advanced-stage ovarian carcinoma (FIGO stage III–IV) were immunostained and evaluated for staining pattern, extent, and intensity. Patients were divided in two groups based on disease outcome. Long-term survivors (21 patients) and short-term survivors (24 patients) were defined using a double cutoff of 36 months for disease-free survival (DFS) and 60 months for overall survival (OS). Mean follow-up period was 70 months. The mean values for DFS and OS were 109 and 125 months for long-term survivors, as compared to 3 and 21 months for short-term survivors, respectively. Results. Comparison of all primary and metastatic lesions showed upregulation of γ-catenin protein expression in the latter ( P = 0.05). When segregated according to disease outcome, the expression of all studied proteins, with the exception of EGFR, was more diffuse in tumors of short-term survivors. The presence of cytoplasmic staining for c-erbB-2 was associated with poor survival in the entire cohort ( P = 0.007), as well as in primary tumors alone ( P = 0.003), in survival analysis. Similar results were seen in the evaluation of primary tumors for γ-catenin ( P = 0.002). Conclusions. γ-Catenin, and possibly c-erbB-2, are valid markers of poor survival in advanced-stage ovarian carcinoma.

Alterations in cellular glycosylation may play a key role in metastatic behaviour of tumour cells. We studied three metastatic cell lines, LOX (malignant melanoma), FEMX (malignant melanoma) and MA-11 (mammary carcinoma). These cell lines have a very different metastatic behaviour in vivo, and different glycans have been postulated to be partly responsible for these differences. To further investigate the functional role of carbohydrates, these three cell lines have been treated with tunicamycin, an inhibitor of the biosynthesis of N-glycans and benzyl- alpha-N-acetylgalactosamine (benzyl-alpha-GalNAc; BnGalNAc), an inhibitor of mature O-linked glycans. Various in vitro adhesion and invasion assays were undertaken for functional studies. Tunicamycin significantly inhibited adhesion to laminin, but only slightly affected cell adhesion to collagen IV. The same compound significantly decreased cellular invasiveness through a Matrigel invasion chamber. Moreover, tunicamycin reduced homotypic aggregation of cells. BnGalNAc had generally little effect on cell behaviour in in vitro assay. The effects of the inhibitors were, however, to some extent cell line-specific. We conclude that N-glycans, but probably not mature O-glycans have important in vitro functions in cell adhesion to laminin, cell invasion through Matrigel and cellular aggregation in the studied cell lines. These results support the view that carbohydrates are functionally involved in several steps of the metastatic process.

The aims of this study were firstly, to investigate the expression of E-cadherin complex proteins in ovarian carcinoma cells in serous effusions and in primary and metastatic lesions; and secondly to study the value of these four proteins and calretinin, a mesothelial marker, in the differential diagnosis of ovarian carcinoma cells from reactive mesothelial cells in effusions. Sixty-seven malignant effusions and 97 corresponding primary (n=36) and metastatic (n=61) lesions were immunohistochemically stained for E-cadherin and alpha-, beta-, and gamma-catenin. Staining extent and intensity were scored. Effusion specimens were additionally analysed for calretinin immunoreactivity. Membrane immunoreactivity for E-cadherin and alpha-, beta-, and gamma-catenin was detected on carcinoma cells in the majority of the effusions, but rarely on reactive mesothelial cells (p

Angiogenic factors play a role in tumor growth and spread. The object of this study was to analyze the correlation between mRNA expression of angiogenesis-related genes and disease outcome in advanced-stage ovarian carcinomas. Sections from 66 primary ovarian carcinomas and metastatic lesions from 41 patients diagnosed with advanced stage ovarian carcinoma (FIGO stages III-IV) were evaluated for expression of basic fibroblast factor (bFGF), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) using mRNA In Situ Hybridization (ISH). Patients were divided in two groups based on disease outcome. Long-term survivors (17 patients) and short-term survivors (24 patients) were defined using a double cut-off of 36 months for disease-free survival (DFS) and 60 months for overall survival (OS). Mean follow-up period was 70 months. The mean values for DFS and OS were 116 and 133 months for long-term survivors, as compared to 3 and 21 months for short-term survivors, respectively. Expression of bFGF mRNA, most often intense, was detected in tumor and stromal cells in the majority of cases. Weak expression of IL-8 mRNA was detected in both cell compartments, while VEGF mRNA expression was limited to few cases. Primary tumors displayed higher bFGF and IL-8 mRNA expression. However, these differences did not reach statistical significance (P>0.05). bFGF, IL-8 and VEGF mRNA expression in both tumor and stromal cells was comparable in tumors of long-term and short-term survivors, and showed no correlation with disease outcome in survival analysis (P>0.05). bFGF is the major angiogenic factor expressed in ovarian carcinoma at the mRNA level. mRNA expression of VEGF, bFGF, and IL-8 does not appear to be a predictor of disease outcome in advanced-stage ovarian carcinoma.

Objective: To investigate the prognostic significance of the carbohydrate epitopes H and Ley and their relationship with proliferation and apoptosis. Study Design: Eighty randomly selected patients with T1-T4 oral tongue squamous cell carcinoma (SCC) were studied. Serial sections were cut from diagnostic, formalin-fixed, paraffin-embedded specimens. Methods: Sections were stained immunohistochemically for H antigen and Ley. Results: Expression of H antigen was associated positively with Ley expression (P = .0001). Expressions of H antigen or Ley correlated with the proliferative markers Ki67 (P = .0442 and P = .0003, respectively) and pAgNOR >1 (P = .0674 and P = .0047, respectively), but not with apoptotic markers such as Bax expression or the apoptotic index (AI). Tumors that expressed H antigen and high levels of Ley (>50%) had a poor prognosis (P = .0006 and P = .0056, respectively). Combinations of expression of H antigen and Ley, and either proliferative or apoptotic markers revealed an enhanced prognostic potential (P < .0001). The combination of pAgNOR score greater than 1 and H-antigen expression appeared to be the best combination to predict good prognosis. Conclusion: The expression of H antigen and Ley, especially their combination with proliferative or apoptotic markers, has prognostic value in tongue SCC.

Objective. The objective of this study was to evaluate the prognostic significance of clinical and histopathologic factors, including a new grading system focusing on the invasive tumor front. Method. A retrospective analysis of 125 surgically treated patients with squamous cell cervical carcinoma FIGO stage IB was conducted. For each tumor, the degree of keratinization, nuclear polymorphism, pattern of invasion, and degree of lymphoid infiltration at the invasive tumor front were graded and given scores between 1 and 4. Results. Clinical tumor size, depth of invasion, and grading of the invasive front had prognostic significance in multivariate analysis, while lymph vascular space involvement, lymph node status, and grade of differentiation did not. Based on clinical tumor size, depth of invasion, and grading of the invasive tumor front, patients could be separated into three groups: One group with minimal risk of recurrence (5-year disease-free survival (DFS) of 100%) consisting of 24% of the patients, an intermediate group with a fairly low risk of recurrence (5-year DFS of about 92%), and a high risk group with a 5-year DFS of 45%. This latter group contained 26% of the patients with 78% of all relapses occurring in the total group of patients. The invasive tumor front grading was reliably reproducible, with inter- and intraobserver agreement of 79 to 87% and κ values of 0.47 to 0.66. Conclusion. Clinical tumor size, depth of invasion, and grading of the invasive tumor front were the main predictors of prognosis in patients with stage IB squamous cell cancer of the cervix.

Keratoacanthoma and squamous cell carcinoma are common skin tumors, especially in immunosuppressed transplant recipients, but the distinction between these two types of epidermal neoplasia may be difficult. Sialyl-Tn (Sia-GalNAc-O-Ser/Thr) is a cell surface carbohydrate associated with hyperplasia in squamous epithelium, and correlated with poor prognosis in several human adenocarcinomas. Twenty-seven keratoacanthomas and 29 cutaneous squamous cell carcinomas were examined for the expression of sialyl-Tn and of the Ki67 epilope, the latter a marker for cell proliferation. By immunohistochemistry, basaloid tumor cells at the periphery of tumor nests showed some degree of sialyl-Tn expression in 16 keratoacanthomas (59%), while only three squamous cell carcinomas (10%) showed sialyl-Tn-positive basaloid tumor cells (p

The object of this study was to analyze the potential association between the expression of MMP-2, MMP-9, MT1-MMP and TIMP-2, and disease outcome in advanced-stage ovarian carcinomas. Sections from 70 paraffin-embedded blocks (36 primary ovarian carcinomas and 34 metastatic lesions) from 45 patients diagnosed with advanced stage ovarian carcinomas (FIGO stages III–IV) were studied using mRNA in situ hybridization (ISH) technique. Patients were divided retrospectively in two groups based on disease outcome. Long-term survivors (21 patients) and short-term survivors (24 patients) were defined using a double cut-off of 36 months for disease-free survival (DFS) and 60 months for overall survival (OS). Mean follow-up period for patients that were diagnosed with advanced-stage carcinoma was 70 months. The mean values for DFS and OS were 109 and 125 months for long-term survivors, as compared to 3 and 21 months for short-term survivors, respectively. Intense mRNA signals were detected more frequently in tumor cells of short-term survivors with use of all four probes. Comparable findings were observed in peritumoral stromal cells with ISH for MMP-2, MMP-9 and TIMP-2 mRNA. Notably, primary tumors with intense mRNA signal for TIMP-2 (No = 14) were uniformly associated with a fatal outcome. In univariate analysis of primary tumors, mRNA levels of TIMP-2 in stromal cells (P = 0.0002), as well as for MMP-9 (P = 0.012) and TIMP-2 (P = 0.02) in tumor cells, correlated with poor outcome. In univariate analysis of metastatic lesions, mRNA levels of TIMP-2 in stromal cells (P = 0.031), as well as for MMP-2 (P = 0.027) and MT1-MMP (P = 0.008) in tumor cells, correlated with poor outcome. Interestingly, the presence of MT1-MMP in stromal cells correlated with longer survival (P = 0.025). In a multivariate analysis of ISH results for primary tumors, TIMP-2 levels in stromal cells (P = 0.006) and MMP-9 levels in tumor cells (P = 0.011) retained their predictive value. We conclude that MMP-2, MMP-9, MT1-MMP and TIMP-2 are valid markers of poor survival in advanced-stage ovarian carcinoma.

Background Small oral squamous cell carcinomas (OSCC) are characterised by a highly variable prognosis. New prognostic factors are needed to give patients the optimum treatment. Budding is defined as a small group of tumour cells or buds with 1–5 cells at the tumour to stroma border, and is accepted as a prognostic factor in colorectal cancer. The purpose of this study was to evaluate if tumour budding can be a prognostic factor for OSCCs. Methods Tumour-tissue from 58 patients with primary, small OSCC, at stages T1/T2, N0, and M0, was collected from the ENT department, Rikshospitalet, Oslo. After diagnosis, patients were primarily treated by surgery and 62% with postoperative radiotherapy. Follow-up information was obtained from their medical records. Budding was detected by immunohistochemistry using anti-pan-keratin antibodies, and tumour budding was scored in the most invasive part of the tumour, registrating the field with most buds (200× magnification). Findings were correlated with treatment and follow-up information. Findings All the carcinomas stained positively for pan-keratin. The tumours were split into two groups: low-budding ( Interpretation Tumour budding in the tumour front was detected in most of the OSCCs, and a high number of buds were associated with a higher frequency of recurrent disease. This indicates a possible clinical value for use of such scoring.

Reactivity of the N-acetylgalactosamine-binding Helix pomatia agglutinin (HPA) in tumours has been associated with poor prognosis and metastasis development. In our LOX/FEMX-I human melanoma model, the binding of HPA correlates with experimental lung metastasis formation in athymic nude mice. In the present study, the metastatic potential of 2 human melanoma cell lines (LOX and FEMX-I) was assessed in relation to carbohydrate and invasive phenotype. Immunocytological and invasion assays highlighted significant differences between these 2 cell lines. Immuno-cytochemical analysis confirmed the widespread expression of HPA-binding glycoconjugates on LOX but not FEMX-I cells. One of these HPA-binding glycoconjugates, the Tn antigen, was expressed highly on the surface of LOX cells but only weakly in the cytoplasm of FEMX-I cells. The sialyl Tn antigen was expressed in FEMX-I but not in LOX cells. There was no difference between the cell lines in adhesion/rate of trapping in athymic nude mouse lung tissues. In Matrigel invasion assays, LOX cells demonstrated an invasion potential more than 6 times greater than that observed with FEMX-I cells. Matrigel invasion of LOX cells was inhibited after incubation with HPA (89%) compared to controls with HPA and GalNAc blocking sugar or without HPA (p < 0.0005 at 5 df). In contrast, there was no inhibitory effect with the anti-Tn antibody IE3. Invasion of FEMX-I cells was not affected by the lectin and the IE3 antibody. Immuno-cytochemical analysis revealed expression of the terminal galactose- and polylactosamine-binding lectin galectin 3 (Mac-2) in these melanoma cell lines. Expression of both the lectin and its receptor may be a contributory feature in the pulmonary invasion of LOX melanoma cells. Overall, our findings suggest that HPA-binding glycoconjugates other than the alphaGalNAc-O-Ser/Thr of the Tn antigen may be important in the extracellular matrix invasion of LOX melanoma cells.

Background The prognostic significance of the invasive tumor front in squamous cell carcinomas has recently been recognized. The aim of the present study was to investigate possible molecular mechanisms underlying the significance of this area in oral squamous cell carcinomas. Methods We used immunohistochemical and molecular genetic techniques to investigate whether p53 alterations at the invasive tumor front could determine the aggressiveness of oral cancers. Results p53 Overexpression was detected in 52% to 56% (four different p53 antibodies) of 100 carcinomas studied. The concordance rate between results of immunohistochemistry and genetic analysis was 60%. No correlation was found between p53 status of the tumors and clinicopathologic parameters analyzed statistically. Conclusions p53 Alterations have no prognostic impact in oral squamous cell carcinomas and apparently do not represent a molecular basis for the biologic significance of the invasive tumor front. The detection of discordant p53 aberrations between primary and second primary carcinomas in some patients provide evidence for their independent origin, with possible impact on prevention and therapy. © 1998 John Wiley & Sons, Inc. Head Neck20: 8–15, 1998.

Purpose: Nuclear texture reflects the overall structures of the chromatin organization. We recently reported the principles and prognostic importance of image analysis of nuclei from metastatic prostate cancer. Immunohistochemical up regulation of the adhesion molecule sialyl Lewisx is also reported to be a prognostic parameter. Presently we analyzed statistically the prognostic impact of these 2 new parameters compared to well-known clinical parameters in metastatic prostate cancer.Materials and Methods: Prognostic factors, such as sedimentation rate, alkaline and acid phosphatases, hemoglobin, testosterone, performance status, pain due to metastasis, T category, histological grade and patient age, were included in a multivariate Cox proportional hazards regression analysis based on 262 patients from the Scandinavian Prostatic Cancer Group Study-2. Extent of bone lesions, deoxyribonucleic acid ploidy, texture analysis and sialyl Lewisx molecules based on subsets of these 262 patients were also an...

Summary Reportedly, the high density of vessels adjacent to various tumours is associated with increased tendency to metastasis and poor prognosis. In contrast, for other cancers a high vessel density is correlated with a good response to radiation therapy, and thus a good prognosis. In this study we measured the vessel density in 53 small glottic SCC (T1N0 and T2N0), treated with radiation (66-70 Gy). The blood vessels were visualised by an immunohistochemical staining method (ABC), using a primary antibody BE2 which reacts with blood group H antigen on endothelial cells. The intra- and interobserver reproducibility of the vessel counting was good (kappa = 0.78). We found a significant correlation between low vascular density and increased risk of recurrent disease (p=0.0158). Cox multivariate analysis showed that both vascular density and T-status were significant prognostic factors, p=0.0036 and p=0.0152 respectively.

Cell migration is a necessary part of malignant invasiveness. Oral squamous cell carcinomas (OSCC) have a great tendency for local invasive growth. We have investigated signalling pathways involved in cell migration induced by epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in OSCC cells and examined the effects of various experimental and clinically approved anti-tumour signal inhibitors on the migratory activity.Migration was studied in three human OSCC cell lines, using a scratch wound assay in vitro and time-lapse cinematography. Specific phosphorylation of signalling proteins was assessed by Western blotting.In the E10 cell line, EGF and HGF induced phosphorylation of EGF receptor (EGFR) and Met, respectively, phosphorylation of ERK1/2, p38 and Akt, and dose-dependent activation of cell migration. Addition of the EGFR-specific inhibitors cetuximab (antibody) or gefitinib (tyrosine kinase blocker) abolished cell migration elicited by EGF. Similarly, a Met kinase inhibitor (SU11274) blocked HGF-induced cell migration. Furthermore, when three cell lines were treated with blockers of the MEK/ERK, p38 or the PI-3 kinase/Akt pathways, the migratory response to both EGF and HGF was inhibited, but to varying degrees. Notably, in E10 and D12 cells, HGF-induced migration was particularly sensitive to PI-3 K-inhibition, while in C12 cells, both HGF- and EGF-induced migration were highly sensitive to p38-blockade.The results demonstrate that the MEK/ERK, p38 and PI-3 kinase pathways are all involved in mediating the increased migration in OSCC cell lines induced by EGF and HGF, but their relative importance and the effects of specific signal inhibitors differ.

J Oral Pathol Med (2012) 41: 547–558 Background: Cell migration is a necessary part of malignant invasiveness. Oral squamous cell carcinomas (OSCC) have a great tendency for local invasive growth. We have investigated signalling pathways involved in cell migration induced by epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in OSCC cells and examined the effects of various experimental and clinically approved anti-tumour signal inhibitors on the migratory activity. Methods: Migration was studied in three human OSCC cell lines, using a scratch wound assay in vitro and time-lapse cinematography. Specific phosphorylation of signalling proteins was assessed by Western blotting. Results: In the E10 cell line, EGF and HGF induced phosphorylation of EGF receptor (EGFR) and Met, respectively, phosphorylation of ERK1/2, p38 and Akt, and dose-dependent activation of cell migration. Addition of the EGFR-specific inhibitors cetuximab (antibody) or gefitinib (tyrosine kinase blocker) abolished cell migration elicited by EGF. Similarly, a Met kinase inhibitor (SU11274) blocked HGF-induced cell migration. Furthermore, when three cell lines were treated with blockers of the MEK/ERK, p38 or the PI-3 kinase/Akt pathways, the migratory response to both EGF and HGF was inhibited, but to varying degrees. Notably, in E10 and D12 cells, HGF-induced migration was particularly sensitive to PI-3 K-inhibition, while in C12 cells, both HGF- and EGF-induced migration were highly sensitive to p38-blockade. Conclusion: The results demonstrate that the MEK/ERK, p38 and PI-3 kinase pathways are all involved in mediating the increased migration in OSCC cell lines induced by EGF and HGF, but their relative importance and the effects of specific signal inhibitors differ.

Alterations of cell-surface carbohydrates are important for the metastatic behaviour of human carcinomas. Follicular and papillary thyroid carcinomas clinically show a very different metastatic pattern and represent a good model for studies of the metastatic process. We examined the expression of various Lex-related carbohydrates in 30 primary papillary, 20 primary follicular and 15 primary anaplastic thyroid carcinomas by means of immunohistochemistry. Six metastases from papillary and five from follicular carcinomas were also examined. Morphologically normal thyroid epithelium did not express any of the type 2 carbohydrates. Papillary carcinomas were immunoreactive for several type 2 carbohydrates, including sialyl-Lewis X, in contrast to follicular and anaplastic carcinomas. The metastases showed no significant differences in expression of carbohydrates compared with the primary tumours. We hypothesize that the up-regulation of sialyl-Lewis X and some other related carbohydrates in papillary carcinomas is of importance for the clinical behaviour of these tumours.

Recent studies indicate that carbohydrate structures are involved in various endothelial functions such as inflammatory processes, adhesion of metastatic cells to endothelium and endothelial differentiation. In this paper we report the endothelial expression of various blood-group-related carbohydrate structures in normal oral mucosa using 15 different monoclonal antibodies reacting with type 1, 2 or 3 carbohydrate chains. Twenty biopsies, including normal oral mucosa, from secretor individuals comprised nine blood group O, nine A, one B and one AB. Endothelial staining was compared with epithelial staining in the same biopsies. Five blood-group-related carbohydrate antigens were detected on endothelial cells. The H type 2 antigen, which is the precursor of A, B and AB antigens, has previously been believed to be a universal marker for endothelial cells. All blood group O individuals (n = 9) showed strong H antigen staining in the endothelium of most vessels. However, of blood group A, B and AB individuals (n = 11), four showed heterogenous H antigen staining. In addition, we found that six out of ten blood group A or AB individuals, who expressed A or A and B antigens on spinous squamous cells and glandular epithelium, showed either heterogenous or no staining for these structures on their endothelial cells. It is concluded that there are differences between the biosynthesis of blood-group-related carbohydrate antigens in oral endothelium and epithelium.

Met, the hepatocyte growth factor receptor, is important in transducing signals for tumour growth and metastasis. The aim of this study was to examine the pattern of Met expression and its value as a prognostic factor in oral squamous cell carcinomas (OSCCs). The material consisted of 53 OSCCs and five healthy controls from normal oral mucosa supplied with cell lines, 10 organotypic models supplied with oral cancer cells, and three organotypic models supplied with normal keratinocytes. Met protein expression was assessed by immunohistochemistry and western blotting. Met expression was scarce and limited to the basal layer in normal oral mucosa, but was more extensive in the tumours. Cytoplasmic expression of Met was found in the majority of the tumours, and nuclear expression was found in 72%, including a high fraction of the cells located at the invasive front. Organotypic models with normal or malignant oral cells yielded principally similar results as in the mucosa and the cancers, respectively. A smaller amount of Met immunoreactivity was detected, by western blotting, in the nuclear fraction of cultured oral cancer cells. In conclusion, Met was upregulated in OSCCs and was also found in the nucleus. However, Met was not a marker for prognosis in this study.

Brusevold IJ, Husvik C, Schreurs O, Schenck K, Bryne M, Soland TM. Induction of invasion in an organotypic oral cancer model by CoCl2, a hypoxia mimetic. Eur J Oral Sci 2010; 118: 168–176. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci Invasion is a hallmark of malignancy. The aim of this study was to develop an in vitro model that can be used for experimental studies of cancer cell invasion. The organotypic oral cancer model was constructed by growing oral squamous cell carcinoma (OSCC) cells on a collagen matrix in which normal human fibroblasts were incorporated. Immunohistochemical staining of the model showed that the expression of invasion-related molecules such as phosphorylated extracellular signal-regulated kinases 1 and 2 (p-ERK1/2), cyclooxygenase-2 (COX-2), p75NTR, and hepatocyte growth factor receptor (Met) was similar to that seen in OSCC. Treatment of the model with cobalt chloride (CoCl2) to mimic hypoxic conditions increased cancer cell invasion, defined as the appearance of cancer cell islands protruding into the matrix. Models treated with CoCl2 showed increased expression of p75NTR and laminin-5 in the cancer cells, and a more pronounced fragmentation of collagen IV in the basal membrane area, in contrast to models that were left untreated. The results indicate that the present model is well suited for studies on cancer cell invasion in the matrix and that the addition of CoCl2 on day 3 of the experiment is indicated because it markedly increases the invasion and improves the model.

Several recent studies have indicated that cells at the invasive tumour margins often are different from cells within other parts of various human cancers. In this work, we have studied all squamous cell carcinomas of the floor of the mouth registered in Norway during the years 1963-1972 (N = 96). Borderline cases and cases given no treatment were excluded. Of the remaining 79 cases, biopsy specimens acceptable for histological grading were obtained from 61 patients. Only the most invasive margins of the tumours were histologically graded independently by two pathologists according to a multifactorial grading system. The results confirmed our previous findings that grading of invasive tumour margins is an independent prognostic factor in Cox's multivariate survival analysis (P less than 0.01). Inter-observer agreement was calculated by kappa statistics, and good agreement was obtained (kappa = 0.63). Neither agreement nor prognostic value was improved after calibration of the pathologists. Conventional Borders' grading of the whole biopsy had no prognostic value (P less than 0.38). We conclude that invasive cell grading may be of value for treatment planning of oral cancers, and that further studies of the deep, invasive parts of oral and other cancers are needed in order to obtain a better understanding of tumour cell invasion and metastasis.

The keratin expression pattern in oral stratified epithelium is related to the cellular differentiation level. The normal pattern shows the keratin pair K5 and K14 in the stratum basale whereas K1 and K10, or K4 and K13, are the two pairs associated with differentiating suprabasal cells. Monoclonal and polyclonal antibodies to individual keratins (K10, K13 and K14) were used in a two-color immunofluorescence staining method to study their coexpression in single cells. Altered keratin expression in premalignant and malignant lesions indicated abnormal differentiation. Monospecific keratin antibodies were suggested to be useful for evaluation of epithelial differentiation changes in oral dysplasias and oral squamous cell carcinomas.

It has been suggested that epithelial cyclooxyge-nase-2 (COX-2) promotes oral carcinogenesis and carcinoma malignancy through increased prostaglandin E2 (PGE2) production. Although oral squamous cell carcinomas (OSCC) often express COX-2, they may also produce PGE2 in a COX-1-dependent manner. We used 6 isolated cell lines to investigate which COX isoforms OSCC may use for PGE2 production. COX-1 and -2 expression patterns divided the 6 OSCC cell lines into 3 distinct groups: both COX isoforms low, only COX-1 high, or both COX isoforms high. Multicolor immunohistofluorescence staining confirmed the COX-expression profiles in organotypic 3D cultures and the COX-2 dominance in OSCC tumors. Epidermal growth factor (EGF) stimulation induced COX-2 (but not COX-1) expression and increased PGE2 production, which was attenuated by COX-2 (but not COX-1) specific inhibition or siRNA-mediated COX-2 gene knockdown. Thus, PGE2 production in OSCC cell lines was COX-2-dependent.

Because blood groups and hemoglobin concentration have been associated with the risk of the development of some cancers, this study evaluated the significance of ABO and Rhesus blood groups and hemoglobin concentration as prognostic factors in oral squamous cell carcinoma (SCC). The authors examined all registered primary SCC of buccal and maxillary alveolar mucosa in the Norwegian population between 1963 and 1972. The biopsy specimens from these patients were reevaluated and borderline cases excluded. The remaining 111 cases were included in the study, and features recorded on first admission were included in the survival analyses. ABO and Rhesus blood groups were found in 99 of these patients. Multivariate survival analysis showed that tumor size, hemoglobin concentration, stage, and Rhesus blood groups were significant prognostic factors, but sex, age, treatment, duration of symptoms, ABO blood groups, and clinical appearance of the tumors were not. The prognostic value of Rhesus blood groups and hemoglobin concentration has not been previously reported for oral SCC.

Recent findings of prognostic value for oral squamous cell carcinomas (OSCCs) which may supplement clinical staging are reviewed. Many reports show that histopathologic grading, measurements of tumor-thickness and DNA-content have independent prognostic value and may thus be of clinical value. Features regarding cells at the invading margins of the tumors are probably of higher prognostic value than features within other parts of the tumors. Reportedly, various other cellular and serum markers have prognostic associations worthwhile further research.

Histomorphological grading at the invasive front of oral squamous cell carcinomas (OSCCs) may provide useful prognostic information. In the present study, we investigated the presence and prognostic value of activated phosphorylated extracellular signal-regulated kinases 1 and 2 (p-ERK1/2) and cyclo-oxygenase-2 (COX-2) both at the invasive front and in central/superficial parts of OSCCs.Using immunohistochemistry, we assessed the presence of p-ERK1/2 and COX-2 in 53 early stage OSCCs. Clinical data were recorded prospectively. The end point was disease-free survival.p-ERK1/2 staining was present in almost all tumours. The staining was mostly nuclear in the cells of the invasive front and either nuclear or nuclear/cytoplasmic in central/superficial tumour parts. COX-2 was observed in almost all tumours (98%) and the staining was often restricted to focal areas. Most tumours were COX-2 negative at the invasive front. The lowest P-value in survival analyses was P = 0.06 for p-ERK1/2 at the invasive front. COX-2, the histomorphological grading systems and TNM stage were of no prognostic value.p-ERK1/2 was present in almost all tumours and p-ERK1/2 may be a prognostic marker at the invasive front of OSCCs. In early stage OSCCs, most tumours did not express COX-2 at the invasive front.

1. Sudbø J. Kjemoprevensjon av munnhulekreft. Nor Tannlegeforen Tid 2003; 113 (7): 320 – 5. 2. Sudbø J. Forskningsprosjekt: Pasienter med høy risiko for munnhulekreft ønskes henvist. Nor Tannlegeforen Tid 2003; 113 (7): 334 – 5. 1. Sudbø J, Sondresen JE, Bryne M. Nytten av å lete etter hvite flekker i munnhulen. Nor tannlegeforen Tid 2001; 111: 920 – 3.

Given the increase in the age distribution of the population, an increase in cancer incidence rates are to be expected. Oral cancer is a disfiguring disease that continues to increase in incidence, particularly in the young, and to an extent that cannot be fully explained by increased exposure to known risk factors. Despite extensive research on treatment modalities towards oral cancer, the 5-year survival rate of this disease has not been improved over the last 4-5 decades. These facts strongly favour chemoprevention-systemic medication to revert, stop, or delay the carcinogenic process-as an approach to treating oral cancer. A chemopreventive approach to oral cancer most likely should encompass a combination of drugs targeting metabolic pathways relevant to oral carcinogenesis. Candidate drugs are retinoids and selective inhibitors of cyclooxygenase-2, epidermal growth factor receptor (EGFR), and peroxisome proliferator activated receptors (PPARs). Chemopreventive trials so far have used surrogate intermediate biomarkers as measurement of treatment effect. However, the efficiency of any drug for chemopreventive use should be assessed through a prospective randomized trial and evaluated by the only definitive end point for prevention of cancer, the incidence rates of new carcinomas.

Emerging data indicate a link between genetic instability and up-regulation of cyclooxygenase-2 (COX-2). To see if individuals at high risk of oral cancer are candidates for treatment with selective COX-2 inhibitors (coxibs), levels of COX-2 expression in healthy, premalignant and cancerous oral mucosa were compared with the occurrence of DNA ploidy status as a genetic risk marker of oral cancer. COX-2 gene product was evaluated immunohistochemically in 30 healthy persons, in 22 patients with dysplastic lesions without previous or concomitant carcinomas, and in 29 patients with oral carcinomas. The immunohistochemical findings were verified by western blotting. COX-2 expression was correlated to DNA content as a genetic risk marker of oral cancer. COX-2 was up-regulated from healthy to premalignant to cancerous oral mucosa. Thus, COX-2 expression was found in 1 case of healthy oral mucosa (3%). All specimens from healthy mucosa had a normal DNA content. In patients with premalignancies. In 29 patients with oral carcinomas, cyclooxygenase-2 expression was observed in 26 (88%), and aneuploidy was observed in 25 cases (94%, P=0.04). Notably, of 22 patients with dysplastic lesions, COX-2 was exclusively expressed in a subgroup of nine patients (41%) identified to be at high risk of cancer by the aberrant DNA content of their lesions. Seven of these patients were followed for 5 years or more. An oral carcinoma developed in six of them (85%; P=0.02). These findings emphasize the need to determine whether coxibs can reduce the risk of oral cancer in patients with high-risk precancerous lesions.

We compared the efficiency of immunophenotyping using flow cytometry (FCM) and a combination of morphologic and immunocytochemical studies for detecting malignant cells in 92 effusions. Cytologic results were as follows: carcinoma cells, 43 specimens; benign, 42 specimens; suggestive of nonepithelial malignancy, 7 specimens. After immunocytochemical analysis, 5 benign specimens were reclassified as malignant and 4 malignant epithelial specimens as benign. With FCM, cells positive for Ber-EP4, B 72.3, AH6, and HB-TN were detected in 28 to 36 (64%-82%) of 44 carcinomas but only 2 to 12 (5%-29%) of 41 benign specimens. Significant association was seen for coexpression. Ber-EP4 and AH6 were the most sensitive; Ber-EP4 was the most specific. The presence of cells positive for 3 of 4 markers strongly suggested malignancy (34/44 carcinoma specimens [77%]; 3/41 reactive specimens [7%]). The presence of cells positive for all 4 markers was diagnostic of malignancy (17/44 malignant specimens [39%]; 0/41 reactive effusions [0%]). FCM and immunocytochemical resultsfor Ber-EP4 expression showed excellent association. FCM is a powerful tool for diagnosing difficult effusions and can quantify coexpression of various markers in fresh specimens. By using established cellular markers coupled with biological markers, FCM also has great promise for experimental purposes.

This study evaluated the prognostic importance of a new grading system focusing on the invasive tumor front, DNA profile, and the proliferation marker MIB-1. A complete geographic series of 172 women treated with radical hysterectomy (Wertheim-Meigs) for FIGO stage I-II cervical carcinomas was the target population. The analyses were performed on 141 (82%) squamous cell carcinomas of the complete series. During the period of observation (mean 222 months), 17 recurrences (12.1%) were encountered. Prognostic factors for disease-free survival were lymph node status (P0.000001), radical surgical margins (P = 0.00004), and tumor size (P = 0.002). The complete score of the invasive front grading system (IFG), and the individual scores of two variables-pattern of invasion and host response-were all significantly (P = 0.002, P = 0.007, P = 0.0001) associated with pelvic lymph node metastases. Host response was the single most important factor in the IFG system, and it was superior to the complete score in predicting lymph node metastases. The total IFG score was also a significant (P = 0.003) prognostic factor for disease-free survival. DNA ploidy, S-phase fraction, and MIB-1 expression were nonsignificant factors in predicting pelvic lymph node metastases and disease-free survival of the patient. The IFG in the original or modified versions could predict low- and high-risk groups of tumors and therefore be of value in treatment planning for these patients.

Angiogenic factors are involved in tumor growth and spread. The aim of this study was to evaluate the expression of angiogenesis-related genes in malignant serous effusions of patients with advanced-stage (FIGO stage III and IV) ovarian carcinoma. In addition, to compare the results for carcinoma cells in effusions with corresponding primary tumors and metastatic lesions, and analyze their prognostic role. Sections from 66 effusions and 90 primary and metastatic lesions from 62 ovarian and primary peritoneal carcinoma patients, were evaluated for expression of basic fibroblast factor (bFGF), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) using mRNA in situ hybridization (ISH). Protein expression was evaluated in a subset of specimens using immunohistochemistry (IHC). ISH results were correlated with clinical parameters. In both effusions and solid tumors, bFGF mRNA was the most commonly expressed factor (93% of effusions and 95% of solid tumors) followed by IL-8, while VEGF was expressed in a minority of the specimens (P0.001 for bFGF vs. IL-8 and VEGF). In solid tumors, angiogenic mRNA expression was seen in both tumor and stromal cells in the majority of positive cases. ISH results did not differ in primary and metastatic tumors. However, carcinoma cells in effusions showed down-regulated expression of VEGF, when compared with both primary tumors (P = 0.029) and metastases (P = 0.015). IL-8 showed a similar down-regulation in effusions, when compared with metastases (P = 0.005). IHC showed excellent agreement with mRNA findings on protein level. In the study of clinico-pathologic parameters, IL-8 mRNA expression in effusions was associated with higher tumor grade (P = 0.044). Angiogenic gene expression in effusions showed no correlation with patient age, previous treatment, residual tumor size, FIGO stage or disease outcome in survival analysis (P0.05). Peritoneal and pleural effusions showed similar expression patterns. In conclusion, bFGF is the major angiogenic factor expressed in ovarian carcinoma at the mRNA level. It is highly expressed in both solid tumors and serous effusions, while IL-8 and VEGF are down regulated in carcinoma cells in effusions, possibly due to the lack of interaction with stromal cells. mRNA expression of VEGF, bFGF, and IL-8 does not appear to be a predictor of disease outcome in advanced-stage ovarian carcinoma. Carcinoma cells in pleural and peritoneal effusions show a similar metastatic expression profile, in agreement with our previous findings, supporting the true metastatic nature of ovarian carcinoma cells in ascites.

Ets-1 proto-oncogene is a transcription factor with a role in the activation of metastasis-associated molecules. We recently found that Ets-1 mRNA expression in solid tumors is a marker of poor prognosis in ovarian carcinoma. The objective of this study was to compare the expression of Ets-1 mRNA in effusions and primary and metastatic tumors of serous ovarian carcinoma patients and to evaluate its prognostic role in effusions. Sections from 67 malignant effusions and 90 primary and metastatic lesions were evaluated for expression of Ets-1 using mRNA in situ hybridization. Expression of Ets-1 mRNA was detected in carcinoma cells in 24 of 67 (36%) effusions. Expression in cancer cells was similar in peritoneal and pleural effusions. In solid lesions Ets-1 expression was detected in both tumor cells and stromal cells in 34 of 90 (38%) lesions. Ets-1 expression in tumor cells showed a strong association with that of stromal cells (p