SET overexpression decreases cell detoxification efficiency: ALDH2 and GSTP1 are downregulated, DDR is impaired and DNA damage accumulates

Alcohol and tobacco consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Aldehyde dehydrogenase 2 (ALDH2) and glutathione Stransferase pi 1 (GSTP1) are important enzymes for cellular detoxification and low efficiencies are implicated in cancer. We assessed the potential role of SET protein overexpression, a histone acetylation modulator accumulated in HNSCC, in gene regulation and protein activity of ALDH2 and GSTP1. SET was knocked down in HN13, HN12 and Cal27, and overexpressed in HEK293 cells; ethanol and cisplatin were the chemical agents. Cells with SET overexpression (HEK293/SET, HN13 and HN12) showed lower ALDH2 and GSTP1 mRNA levels and trichostatin A increased them (real-time PCR). Ethanol upregulated GSTP1 and ALDH2 mRNAs, whereas cisplatin upregulated GSTP1 in HEK293 cells. SET-chromatin binding revealed SET interaction with ALDH2 and GSTP1 promoters, specifically via SET NAP domain; ethanol and cisplatin abolished SET binding. ALDH2 and GSTP1 efficiency was assessed by enzymatic and comet assay. A lower ALDH2 activity was associated with greater DNA damage (tail intensity) in HEK293/SET compared with HEK293 cells, whereas HN13/siSET showed ALDH2 activity higher than HN13 cells. HN13/siSET cells showed increased tail intensity. Cisplatin-induced DNA damage response showed negative relationship between SET overexpression and BRCA2 recruitment. SET downregulated repair genes ATM, BRCA1 and CHEK2 and upregulated TP53. Cisplatin-induced cell-cycle arrest occurred in G0/G1 and S in HEK293 cells, whereas HEK293/SET showed G2/M stalling. Overall, cisplatin was more cytotoxic for HN13 than HN13/siSET cells. Our data suggest a role for SET in cellular detoxification, DNA damage response and genome integrity.

Clinicopathological And Immunohistochemical Evaluation Of Oral And Oropharyngeal Squamous Cell Carcinoma In Chilean Population.

In oral and oropharyngeal squamous cell carcinoma (OCSCC and OPSCC) exist an association between clinical and histopathological parameters with cell proliferation, basal lamina, connective tissue degradation and surrounding stroma markers. We evaluated these associations in Chilean patients. A convenience sample of 37 cases of OCSCC (n=16) and OPSCC (n=21) was analyzed clinically (TNM, clinical stage) and histologically (WHO grade of differentiation, pattern of tumor invasion). We assessed the expression of p53, Ki67, HOXA1, HOXB7, type IV collagen (ColIV) and carcinoma-associated fibroblast (α-SMA-positive cells). Additionally we conducted a univariate/bivariate analysis to assess the relationship of these variables with survival rates. Males were mostly affected (56.2% OCSCC, 76.2% OPSCC). Patients were mainly diagnosed at III/IV clinical stages (68.8% OCSCC, 90.5% OPSCC) with a predominantly infiltrative pattern invasion (62.9% OCSCC, 57.1% OPSCC). Significant association between regional lymph nodes (N) and clinical stage with OCSCC-HOXB7 expression (Chi-Square test P < 0.05) was observed. In OPSCC a statistically significant association exists between p53, Ki67 with gender (Chi-Square test P < 0.05). In OCSCC and OPSCC was statistically significant association between ki67 with HOXA1, HOXB7, and between these last two antigens (Pearson's Correlation test P < 0.05). Furthermore OPSCC-p53 showed significant correlation when it was compared with α-SMA (Kendall's Tau-c test P < 0.05). Only OCSCC-pattern invasion and OPSCC-primary tumor (T) pattern resulted associated with survival at the end of the follow up period (Chi-Square Likelihood Ratio, P < 0.05). Clinical, histological and immunohistochemical features are similar to seen in other countries. Cancer proliferation markers were associated strongly from each other. Our sample highlights prognostic value of T and pattern of invasion, but the conclusions may be limited and should be considered with caution (small sample). Many cases were diagnosed in the advanced stages of the disease, which suggests that the diagnosis of OCSCC and OPSCC is made late.

Enhanced programmed death 1 (PD-1) and PD-1 ligand (PD-L1) expression in patients with actinic cheilitis and oral squamous cell carcinoma

PD-1 and PD-L1 can be involved in tumor escape, and little is known about the role of these molecules in oral tumors or pre-malignant lesions. In the present study, we investigated the expression of PD-1 and PD-L1 in the blood and lesion samples of patients with actinic cheilitis (AC) and oral squamous cell carcinoma (OSCC). Our results showed that lymphocytes from peripheral blood and tissue samples exhibited high expression of PD-1 in both groups analyzed. Patients with AC presented higher percentage as well as the absolute numbers of CD4(+)PD-1(+) and CD8(+)PD-1(+) lymphocytes in peripheral blood mononuclear cells (PBMC) than healthy individuals, while patients with OSCC presented an increased frequency of CD8(+)PD1(+) in PBMC when compared with controls. On the other hand, increased frequency of CD4(+) and CD8(+) T cells expressing PD-1(+) accumulate in samples from OSCC, and the expression of PD-L1 was intense in OSCC and moderate in AC lesion sites. Lower levels of IFN-gamma and higher levels of TGF-beta were detected in OSCC samples. Our data demonstrate that PD-1 and PD-L1 molecules are present in blood and samples of AC and OSCC patients. Further studies are required to understand the significance of PD-1 and PD-L1 in oral tumors microenvironment.

Watermelon Stomach, Hemorrhagic Pericarditis, Small Cell Carcinoma of the Lung and Synchronous Squamous Cell Carcinoma of the Tongue Base

Based on a case of gastric antral vascular ectasia (watermelon stomach) that was associated with hemorrhagic pericarditis, small cell lung carcinoma with mediastinal lymph node metastases and a synchronous squamous cell carcinoma of the base of the tongue, the authors made a review of the clinical, endoscopic and histopathological aspects of this type of gastropathy, and its association with other diseases, and of the results of its endoscopic therapy. The causes of hemorrhagic pericarditis are considered, emphasizing the necessity to know if the effusion has a malignant etiology. To the best of our knowledge the association of watermelon stomach to small cell lung carcinoma and squamous cell carcinoma of the base of the tongue has not yet been described. Extensive metastases to mediastal lymph nodes are common to small cell lung carcinoma.

Correlation between koilocytes and human papillomavirus detection by PCR in oral and oropharynx squamous cell carcinoma biopsies

The purpose of this study was to compare the histopathological analysis with polymerase chain reaction (PCR) methods to predict the presence of human papillomavirus (HPV) infection in oral squamous cell carcinoma biopsies. Eighty-three paraffin-embedded tissue specimens from patients with oropharynx and mouth floor squamous cell carcinoma were submitted to histopathological analysis under light microscopy, specifically for the determination of the presence of koilocytes. Subsequently, DNA was purified from the same paraffin-embedded specimens and submitted to PCR. Fisher's exact test showed no statistically significant correlation between the two methods. The results suggest that the presence of koilocytes is unreliable for the detection of HPV presence in oral and oropharynx squamous cell carcinoma.

Human papillomavirus infection and p53 protein expression in vulvar intraepithelial neoplasia and invasive squamous cell carcinoma

The etiopathogenesis of vulvar intraepithelial neoplasia (VIN III) and invasive squamous cell carcinoma are largely unknown. Since there are few studies on Brazilian patients, our purpose was to determine the frequency of human papillomavirus (HPV) infection and the expression of p53 in these lesions, and associate them with other factors such as age, morphological subtypes, multicentric and multifocal disease. Thirty-eight cases of VIN III, nine of superficially invasive carcinoma, and 55 of invasive vulvar carcinoma were retrospectively evaluated from 1983 to 1995 for the presence of HPV by immunohistochemistry and in situ hybridization, and for p53 protein expression by immunohistochemistry on paraffin sections. All cases for whom material (slides and paraffin blocks) and clinical data were available were included. HPV and p53 were detected in 57.9 and 21.1% of the VIN III lesions, 33.3 and 66.7% of superficially invasive carcinomas, and 7.3 and 58.2% of invasive squamous cell carcinomas, respectively. HPV infection was associated with younger age in the VIN III and invasive carcinoma groups. In the latter, HPV infection was associated with the basaloid variant. p53 expression rate was higher in superficially invasive and invasive lesions and was not related to HPV infection. Our findings are similar to others and support the hypothesis that there are two separate entities of the disease, one associated with HPV and the other unrelated, with p53 inactivation possibly being implicated in some of the cases.

Classic and molecular cytogenetic analyses reveal chromosomal gains and losses correlated with survival in head and neck cancer patients

Purpose: Genetic biomarkers of head and neck tumors could be useful for distinguishing among patients with similar clinical and histopathologic characteristics but having differential probabilities of survival. The purpose of this study was to investigate chromosomal alterations in head and neck carcinomas and to correlate the results with clinical and epidentiologic variables.Experimental Design: Cytogenetic analysis of short-term cultures from 64 primary untreated head and neck squamous cell carcinomas was used to determine the overall pattern of chromosome aberrations. A representative subset of tumors was analyzed in detail by spectral karyotyping and/or confirmatory fluorescence in situ hybridization analysis.Results: Recurrent losses of chromosomes Y (26 cases) and 19 (14 cases), and gains of chromosomes 22 (23 cases), 8 and 20 (11 cases each) were observed. The most frequent structural aberration was del(22)(q13.1) followed by rearrangements involving 6q and 12p. The presence of specific cytogenetic aberrations was found to correlate significantly with an unfavorable outcome. There was a significant association between survival and gains in chromosomes 10 (P = 0.008) and 20 (P = 0.002) and losses of chromosomes 15 (P = 0.005) and 22 (P = 0.021). Univariate analysis indicated that acquisition of monosomy 17 was a significant (P = 0.0012) factor for patients with a previous family history of cancer.Conclusions: the significant associations found in this study emphasize that alterations of distinct regions of the genome may be genetic biomarkers for a poor prognosis. Losses of chromosomes 17 and 22 can be associated with a family history of cancer.

Correlation between koilocytes and human papillomavirus detection by PCR in oral and oropharynx squamous cell carcinoma biopsies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative analysis of cell proliferation ratio in oral lichen planus, epithelial dysplasia and oral squamous cell carcinoma

Background: Although oral lichen planus has been classified by the World Health Organization (WHO) as a potentially malignant disorder, such classification is still the target of much controversy. Aim: To evaluate the cell proliferation rate in oral lichen planus, comparing it to the rate observed in epithelial dysplasia and oral squamous cell carcinoma, aiming at indications which might indicate the potential for malignant transformation. Material and Methods: Twenty-four cases of each lesion were submitted to the streptoavidin-biotin and AgNOR technique to evaluate the immunohistochemical expression of PCNA and the mean NORs/ nucleus, respectively. Results: Positivity for PCNA was observed in 58.33% of oral lichen planus cases, 83.33% of epithelial dysplasia cases and 91.67% of oral squamous cell carcinoma cases. Chi-squared test showed that the number of positive cases for PCNA was significantly lower in oral lichen planus than in oral squamous cell carcinoma (p<0.05). No significant statistical difference between oral lichen planus and epithelial dysplasia (p>0.05) and between the epithelial dysplasia and oral squamous cell carcinoma (p>0.05) was observed. The mean NORs/ nucleus in oral lichen planus, epithelial dysplasia and oral squamous cell carcinoma were 1.74 +/- 0.32, 2.42 +/- 0.62 e 2.41 +/- 0.61, respectively. Variance analysis (ANOVA) revealed significant statistical difference between oral lichen planus and the other studied lesions (p<0.05). Conclusion: Oral lichen planus cell proliferation rate was less than in oral epithelial dysplasia and oral squamous cell carcinoma which might explain the lower malignant transformation rate.

Comparative analysis of the expression of proliferating cell nuclear antigen, p53, bax, and bcl-2 in oral lichen planus and oral squamous cell carcinoma

Several epidermiologic studies have shown the malignant transformation potential of oral lichen planus; however, this potential is subject of much controversy. To evaluate the expression of proteins related to the cell proliferation and apoptosis processes in oral lichen planus, we compared oral lichen planus with oral squamous cell carcinoma. Twenty-four cases of each lesion were submitted according to streptavidin-biotin technique to evaluate the immunohistochemical expression of proliferating cell nuclear antigen, p53, bax, and bcl-2 proteins. X 2 test showed no statistically significant differences between the expression of p53, bax, and bcl-2 in oral lichen planus and oral squamous cell carcinoma (P > .05). However, the expression of proliferating cell nuclear antigen was significantly lower in oral lichen planus than in oral squamous cell carcinoma (P < .05). No statistically significant differences between the expression of p53, bax, and bcl-2 in oral lichen planus and oral squamous cell carcinoma were observed, which may be an evidence of the potential of malignant transformation of oral lichen planus. (C) 2009 Elsevier B.V. All rights reserved.

Lysyl oxidase expression is an independent marker of prognosis and a predictor of lymph node metastasis in oral and oropharyngeal squamous cell carcinoma (OSCC)

Proteins of the lysyl oxidase (LOX) family are important modulators of the extracellular matrix. However, they have an important role in the tumour development as well as in tumour progression. To evaluate the diagnostic and prognostic value of the LOX protein in oral and oropharyngeal squamous cell carcinoma (OSCC) we performed QRT-PCR and immunohistochemical analysis on two tissue microarrays (622 tissue samples in total). Significantly higher LOX expression was detected in high grade dysplastic oral mucosa as well as in OSCC when compared to normal oral mucosa (P < 0.001). High LOX expression was correlated with clinical TNM stage (P = 0.020), lymph node metastases for the entire cohort (P < 0.001), as well as in the subgroup of small primary tumours (T1/T2, P < 0.001). Moreover, high LOX expression was correlated with poor overall survival (P = 0.004) and disease specific survival (P = 0.037). In a multivariate analysis, high LOX expression was an independent prognostic factor, predicting unfavourable overall survival. In summary, LOX expression is an independent prognostic biomarker and a predictor of lymph node metastasis in OSCC. Moreover, LOX overexpression may be an early phenomenon in the pathogenesis of OSCC and thus an attractive novel target for chemopreventive and therapeutic strategies.

Definitive intensity modulated radiotherapy in locally advanced hypopharygeal and laryngeal squamous cell carcinoma: mature treatment results and patterns of locoregional failure.

PurposeTo assess clinical outcomes and patterns of loco-regional failure (LRF) in relation to clinical target volumes (CTV) in patients with locally advanced hypopharyngeal and laryngeal squamous cell carcinoma (HL-SCC) treated with definitive intensity modulated radiotherapy (IMRT) and concurrent systemic therapy.MethodsData from HL-SCC patients treated from 2007 to 2010 were retrospectively evaluated. Primary endpoint was loco-regional control (LRC). Secondary endpoints included local (LC) and regional (RC) controls, distant metastasis free survival (DMFS), laryngectomy free survival (LFS), overall survival (OS), and acute and late toxicities. Time-to-event endpoints were estimated using Kaplan-Meier method, and univariate and multivariate analyses were performed using Cox proportional hazards models. Recurrent gross tumor volume (RTV) on post-treatment diagnostic imaging was analyzed in relation to corresponding CTV (in-volume, > 95% of RTV inside CTV; marginal, 20¿95% inside CTV; out-volume, < 20% inside CTV).ResultsFifty patients (stage III: 14, IVa: 33, IVb: 3) completed treatment and were included in the analysis (median follow-up of 4.2 years). Three-year LRC, DMFS and overall survival (OS) were 77%, 96% and 63%, respectively. Grade 2 and 3 acute toxicity were 38% and 62%, respectively; grade 2 and 3 late toxicity were 23% and 15%, respectively. We identified 10 patients with LRF (8 local, 1 regional, 1 local¿+¿regional). Six out of 10 RTVs were fully included in both elective and high-dose CTVs, and 4 RTVs were marginal to the high-dose CTVs.ConclusionThe treatment of locally advanced HL-SCC with definitive IMRT and concurrent systemic therapy provides good LRC rates with acceptable toxicity profile. Nevertheless, the analysis of LRFs in relation to CTVs showed in-volume relapses to be the major mode of recurrence indicating that novel strategies to overcome radioresistance are required.

The transcriptional repressor Delta EF1 controls resistance to the EGFR inhibitor erlotinib in human HNSCC lines

Epidermal Growth Factor Receptor (EGFR) overexpression occurs in about 90% of Head and Neck Squamous Cell Carcinoma (HNSCC) cases. Aberrant EGFR signaling has been implicated in the malignant features of HNSCC. Thus, EGFR appears to be a logical therapeutic target with increased tumor specificity for the treatment of HNSCC. Erlotinib, a small molecule tyrosine kinase inhibitor, specifically inhibits aberrant EGFR signaling in HNSCC. Only a minority of HNSCC patients were able to derive a substantial clinical benefit from erlotinib. ^ This dissertation identifies Epithelial to Mesenchymal Transition (EMT) as the biological marker that distinguishes EGFR-dependent (erlotinib-sensitive) tumors from the EGFR-independent (erlotinib-resistant) tumors. This will allow us to prospectively identify the patients who are most likely to benefit from EGFR-directed therapy. More importantly, our data identifies the transcriptional repressor DeltaEF1 as the mesenchymal marker that controls EMT phenotype and resistance to erlotinib in human HNSCC lines. si-RNA mediated knockdown of DeltaEF1 in the erlotinib-resistant lines resulted in reversal of the mesenchymal phenotype to an epithelial phenotype and significant increase in sensitivity to erlotinib. ^ DeltaEF1 represses the expression of the epithelial markers by recruiting HDACs to chromatin. This observation allows us to translate our findings into clinical application. To test whether the transcriptional repression by DeltaEF1 underlines the mechanism responsible for erlotinib resistance, erlotinib-resistant lines were treated with an HDAC inhibitor (SAHA) followed by erlotinib. This resulted in a synergistic effect and substantial increase in sensitivity to erlotinib in the resistant cell lines. Thus, combining an HDAC inhibitor with erlotinib represents a novel promising pharmacologic strategy for reversing resistance to erlotinib in HNSCC patients. ^