Centromere cleavage is a mechanism underlying isochromosome formation in skin and head and neck carcinomas

Centromeric rearrangements, in the form of isochromosomes or whole-arm translocations, are the most common recurrent changes in head and neck and skin carcinomas. Little is known about the mechanisms behind the origin of these chromosome rearrangements. In the present study, one basal cell carcinoma and two squamous cell carcinomas of the head and neck were thoroughly studied by cytogenetic and fluorescence in situ hybridization techniques. All tumors showed intratumor heterogeneity in the form of cytogenetically related subclones (in all tumors) and unrelated clones (in one tumor). Assessment of karyotypic evolution in these tumors suggests that centromeric cleavage is a mechanism giving rise to isochromosomes. A similar mechanism may also be involved in the formation of whole-arm translocations.     

Radiation-induced changes of fibroblasts in the squamous cell carcinoma of the head and neck

The regrowth of mesenchymal tissue (stroma) surrounding the malignant epithelium is an important step in tissue remodelling during and after irradiation. The radiation-induced fibroblastic changes were studied on tissue samples taken before, during and after the radical irradiation of the squamous cell carcinoma of the head and neck. Elongated fibroblasts with large amount of rough endoplasmic reticulum were seen around the tumor epithelium before radiation. The fibrosis increased during irradiation and at the same time the shape of the fibroblasts changed so that they became more triangular and nuclear structures became more prominent together with hyperchromasia. The amount of cell organelles declined although there was a large amount collagen present. Epithelial cells invaded through the basal lamina. In most samples the basal lamina could not be seen at all and the tumor cells were dispersed between stromal elements. On the other hand there were close contacts between epithelial and mesenchymal cells throughout the study in places where the basal lamina was broken, which might indicate epithelio-mesenchymal interaction. Also the connective tissue formed by fibroblasts and collagen might be part of the radiation induced healing and destruction of the tumor cells.     

Radiation-induced circulating miRNA expression in blood of head and neck cancer patients

In recent years, scientists have found evidence confirming the aberrant expression of miRNAs in cancer patients compared to healthy individuals. The growing interest in the identification of non-invasive and specific diagnostic and prognostic molecular markers has identified microRNAs as potential candidates in cancer diagnosis, prognosis and treatment response. In the present study, we have analyzed the expression profile of circulating miR-21, -191 and -421 in peripheral blood of head and neck cancer patients (HNC) to investigate a possible modulation of mRNA levels by radiation and to identify the role of mRNA as biomarkers of cancer prognosis. Results showed a modulation of the microRNA expression at different time points after radiotherapy, suggesting that treatment may influence the release of circulating miRNAs depending also on the time interval elapsed since radiotherapy. The expression levels of miR-21, -191 and -421 were higher in blood of patients treated with radiotherapy alone after 6 months from the end of therapy and high levels of them seemed to correlate with the remission of the disease. The trends shown in this study confirmed that miRNAs could be useful prognosis markers and could provide preliminary data for further evaluation in predicting patients’ response to radiotherapy by developing miRNA-based treatments to improve the sensitivity of cancer cells to radiotherapy.     

Production of granulocyte colony-stimulating factor by head and neck carcinomas

Detectable levels of G-CSF by enzyme-linked immunosorbent assay (ELISA) were found in sera of 4 out of 15 patients with head and neck carcinomas. Also cells prepared from the tumors of these 4 patients secreted G-CSF. The supernatants of cells derived from all 15 patients did not contain granulocyte-monocyte CSF, monocyte CSF, tumor necrosis factor-, transforming growth factor- ₁, epidermal growth factor, interleukin (IL)-1 and IL-6. These findings suggest that leukocytosis in patients with carcinomas might be due to the production of G-CSF by tumor cells.Abbreviations CSF colony stimulating factor - EGF epidermal growth factor - ELISA Enzyme-linked immunosorbent assay - G granulocyte - GM granulocyte-monocyte - IL interluekin - M monocyte - TGF transforming growth factor - TNF tumor necrosis factor     

CK8 correlates with malignancy in leukoplakia and carcinomas of the head and neck

Screening of head and neck carcinoma patients with the proteomics-based AMIDA technology yielded a set of tumour-associated antigens, including the intermediate filament protein cytokeratin 8 (CK8). The expression pattern and specificity of CK8 was compared with those of the established markers pan-cytokeratins and CK13, and with that of the proliferation marker Ki67. Expression of CK8 correlated positively with malignancies of the head and neck areas. CK8 was not expressed in healthy epithelium, except for some rare cases of cells of the basal layer and laryngeal tissue. In contrast, the vast majority of head and neck squamous cell carcinomas and metastases strongly expressed CK8. Interestingly, CK8 de novo expression correlated with dysplastic areas of oral leukoplakic lesions, while hyperplastic leukoplakia remained CK8-negative but strongly panCK and CK13 positive. Thus, CK8 is an attractive marker molecule for a differentiated diagnosis of leukoplakia and head and neck carcinomas, which possesses notedly improved specificity as compared with panCK and CK13.     

Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

Tumor size-dependent elevations of serum gangliosides in patients with head and neck carcinomas

Serum gangliosides were studied in 100 patients with benign or tumoral head and neck lesions. Whereas very few changes over the normal level were noticed in benign cases, 78% of patients with head and neck carcinomas had a significant elevation of serum gangliosides, mostly accounted for by GM3 and GD3. Such an increase was correlated with the observed tumor size, and the level of serum gangliosides returned to normal within one month after surgery. Follow-up of patients showed that further elevations of serum gangliosides were associated with relapses.     

Mean nuclear area of squamous cell carcinomas of the head and neck using image cytometry.

OBJECTIVE: To determine if mean nuclear area (MNA) in squamous cell carcinomas of the head and neck (SCCHN) correlate with the TNM system and histologic grade. STUDY DESIGN: We measured MNA by image cytometry on 74 primary SCCHN. Fify-five had primary surgery, 16 had radiotherapy, and 3 and both as their primary treatment. RESULTS: The mean MNA was 47.85 microm2 (range, 20.5-84.8). Tumor size, nodal status and histologic grade were, respectively: T1 = 13, T2 = 29, T3 = 18, T4 = 14; N0 = 53, N1 = 15, N2 = 5, N3 = 1; 17 = well, 38 = moderate, 19 = poorly differentiated. Spearman rank and Kruskal-Wallis tests for MNA/histologic grade, MNA/tumor size, MNA/nodal status and MNA/site were calculated; only MNA/node was statistically significant (P<.05). CONCLUSION: MNA increases in primary SCCHN as nodal involvement increases. This may reflect that high MNA may be a biologic marker of primary SCCHN with a poorer prognosis.     

Autologous tumor cell killing activity of tumor-associated lymphocytes in patients with head and neck carcinomas

In order to select the most cytotoxic effector cells for adoptive immunotherapy, lymphokine activated killer (LAK) cells, tumor infiltrating lymphocytes (TILs) and autologous mixed lymphocyte tumor cell culture (MLTC) cells derived from peripheral blood mononuclear cells (PBMC) in the same subject with head and neck carcinomas were prepared. The autologous tumor cell killing activity and cell surface phenotypes of each of the three effector cells were studied. MLTC cells cultured with interleukin-2 (IL-2) showed the strongest cytotoxic activity among these three different effector cells. Although TILs had suppressed killing activity immediately after isolation, after successive cultivations with IL-2, a cytotoxic activity against autologous tumor cells stronger than that of LAK cells appeared. Both IL-2 stimulated MLTC cells and TILs showed an enrichment of CD8 positive and CDU negative cells in a CD3 positive subpopulation.Abbreviations CD cluster differentiation - IL-2 interleukin-2 - LA lymphokine activated - LAK lymphokine activated killer - MLTC mixed lymphocyte tumor cell culture - NK natural killer - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Prognostic value of p53 overexpression in head and neck carcinomas: a pilot study

OBJECTIVE: To correlate p53 overexpression in squamous cell carcinoma of the head and neck region with the outcome of treatment. STUDY DESIGN: Twenty-five biopsy-proven squamous cell carcinomas of the head and neck region, locally advanced and untreated, were studied. Before treatment, all patients underwent fine needle aspiration from primary and/or metastatic lesions. Smears were prepared from the aspirate for immunostaining, and p53 overexpression was measured semiquantitatively. All patients received a radical dosage of radiation equivalent to 60 Gy for 6 weeks in 30 fractions from a 6-MV linear accelerator. Local-regional disease control was studied, and the mean follow-up duration was one year. The pretreatment values of p53 overexpression were correlated with the outcome of treatment. RESULTS: Overexpression of p53 was found in 36% patients. At the end of 1 year, 6/9 patients showing overexpression were disease free as compared to 5/16 patients without overexpression. The difference was not significant (chi2 test, P>.05). CONCLUSION: Response to radiation therapy is not dependent on p53 overexpression in squamous cell carcinoma of the head and neck region. However, this was only a pilot study, and a large number of cases are needed to establish the prognostic value of p53 overexpression in locally advanced head and neck squamous cell carcinoma.     

Correlation between nitric oxide and cyclooxygenase-2 pathways in head and neck squamous cell carcinomas

We investigated the interactions between inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) pathways in head and neck squamous cell carcinomas (HNSCCs) and in two carcinoma cell lines. HNSCCs showed an up-regulation of both pathways which were strongly correlated with each other (p=0.02) and with tumor vascularization (p=0.0001 and p=0.008, respectively). In carcinoma cells, Escherichia coli lipopolysaccharide (LPS) and EGF treatment up-regulated both pathways. NOS inhibitor N(G)-monomethyl-L-arginine methyl ester (L-NAME) inhibited this up-regulation. LPS or EGF induced iNOS expression that was not altered by NOS or COX-2 inhibitors. Conversely, LPS or EGF promoted COX-2 expression that was decreased by L-NAME. The NO donor S-nitroso-acetyl-penicillamine (SNAP) up-regulated COX-2 pathway and this effect was reduced by the guanylate cyclase inhibitor methylene blue. Thus, in squamous carcinoma cells, NO increases the activity of COX-2 pathway and this effect is probably mediated by endocellular cGMP level, with potential implications on tumor growth, angiogenesis, and therapy.     

Aberrant glycosylation based on the neo-expression of poly- N-acetyllactosamine structures in squamous cell carcinomas of the head and neck

An immuno- and lectin-histochemical study was performed to investigate the aberrant expression of blood group-related antigens and poly-N- acetyllactosamine structures in squamous cell carcinomas of the maxillary sinus, the larynx, the apipharynx, the hypopharynx, the oral cavity, the parotid gland and the tonsil from 52 patients using monoclonal antibodies against A, B and H antigens, and six lectins, UEA-I, PNA, VVA-B4, PWM, LEA and DSA. In addition, GSA- II staining following endo-·-galactosidase digestion procedure was also applied. A, B and H antigens were expressed in most normal epithelial cells of head and neck organs, and depended on the patient blood type. However, in squamous cell carcinoma, A antigen was not detected in eight out of 25 individuals of blood groups A and AB, although B antigen was consistently expressed in carcinoma cells from all the B and AB individuals. On the other hand, H antigen was expressed in carcinoma cells not only from all blood group O individuals, but from 32 out of 35 individuals of blood groups A, B and AB. T and Tn antigens, which are recognized by PNA and VVA-B4, were strongly expressed in carcinoma cells from 40 and 42 out of 52 individuals respectively. Reactivity with GSA-II staining following endo-·-galactosidase digestion, which recognizes linear poly-N-acetyllactosamine structures, was found in a few malignant cells from 21 individuals. Staining with anti-A, -B and -H monoclonal antibodies and UEA-I lectin was diminished after endo-·-galactosidase digestion in some cases. Lectins specific for poly-N-acetyllactosamine, such as PWM, LEA and DSA, exhibited reactivity in some malignant cells from 30, 22 and 32 out of 52 individuals respectively. These results suggested that the expression of the blood group-related antigens is suppressed and immature carbohydrate chains, that is H, T and Tn antigens, are accumulated in squamous cell carcinomas of the head and neck. The results further suggested that poly-N-acetyllactosamine structures are simultaneously synthesized along with the deletion of A antigen and the accumulation of precursors This revised version was published online in November 2006 with corrections to the Cover Date.     

Cancer of the head and neck.

Cancers of the upper aerodigestive tract, collectively known as head and neck cancers, arise from a multiplicity of sites. In the West, excess tobacco and alcohol consumption are the most important of the known predisposing factors; elsewhere in the world, notably in India and China, the aetiology, pattern of primary sites, and clinical behaviour are different. Clinically these tumours pose exceptional problems in management, and skilled multidisciplinary teams are necessary in order to achieve the highest level of service and research. Historically, surgery and radiotherapy have been the most important treatment modalities; chemotherapy is now increasingly employed but not yet fully established. Successful rehabilitation of patients with head and neck cancers requires access to high quality speech therapists and other support staff with training in functional pharyngeal disorders. Current research efforts are largely directed towards defining the proper role of chemotherapy and assessing the possible advantage of unconventional radiation approaches. In recent years the roles of primary, reconstructive, and salvage surgery have also become better defined. Many patients are suitable for randomisation into ongoing prospective clinical trials which have been specifically designed to address these issues.     

Pretreatment dietary intake is associated with tumor suppressor DNA methylation in head and neck squamous cell carcinomas

Diet is associated with cancer prognosis, including head and neck cancer (HNC), and has been hypothesized to influence epigenetic state by determining the availability of functional groups involved in the modification of DNA and histone proteins. The goal of this study was to describe the association between pretreatment diet and HNC tumor DNA methylation. Information on usual pretreatment food and nutrient intake was estimated via food frequency questionnaire (FFQ) on 49 HNC cases. Tumor DNA methylation patterns were assessed using the Illumina Goldengate Methylation Cancer Panel. First, a methylation score, the sum of individual hypermethylated tumor suppressor associated CpG sites, was calculated and associated with dietary intake of micronutrients involved in one-carbon metabolism and antioxidant activity, and food groups abundant in these nutrients. Second, gene specific analyses using linear modeling with empirical Bayesian variance estimation were conducted to identify if methylation at individual CpG sites was associated with diet. All models were controlled for age, sex, smoking, alcohol and HPV status. Individuals reporting in the highest quartile of folate, vitamin B12 and vitamin A intake, compared with those in the lowest quartile, showed significantly less tumor suppressor gene methylation, as did patients reporting the highest cruciferous vegetable intake. Gene specific analyses identified differential associations between DNA methylation and vitamin B12 and vitamin A intake when stratifying by HPV status. These preliminary results suggest that intake of folate, vitamin A and vitamin B12 may be associated with the tumor DNA methylation profile in HNC and enhance tumor suppression.     

Pretreatment dietary intake is associated with tumor suppressor DNA methylation in head and neck squamous cell carcinomas

Diet is associated with cancer prognosis, including head and neck cancer (HNC), and has been hypothesized to influence epigenetic state by determining the availability of functional groups involved in the modification of DNA and histone proteins. The goal of this study was to describe the association between pretreatment diet and HNC tumor DNA methylation. Information on usual pretreatment food and nutrient intake was estimated via food frequency questionnaire (FFQ) on 49 HNC cases. Tumor DNA methylation patterns were assessed using the Illumina Goldengate Methylation Cancer Panel. First, a methylation score, the sum of individual hypermethylated tumor suppressor associated CpG sites, was calculated and associated with dietary intake of micronutrients involved in one-carbon metabolism and antioxidant activity, and food groups abundant in these nutrients. Second, gene specific analyses using linear modeling with empirical Bayesian variance estimation were conducted to identify if methylation at individual CpG sites was associated with diet. All models were controlled for age, sex, smoking, alcohol and HPV status. Individuals reporting in the highest quartile of folate, vitamin B12 and vitamin A intake, compared with those in the lowest quartile, showed significantly less tumor suppressor gene methylation, as did patients reporting the highest cruciferous vegetable intake. Gene specific analyses identified differential associations between DNA methylation and vitamin B12 and vitamin A intake when stratifying by HPV status. These preliminary results suggest that intake of folate, vitamin A and vitamin B12 may be associated with the tumor DNA methylation profile in HNC and enhance tumor suppression.