Elevated translationally controlled tumour protein promotes oral cancer progression and poor outcome.

BACKGROUND: Translationally controlled tumour protein (TCTP) is a multifunctional protein elevated in multiple cancers. However, studies on its role in oral carcinogenesis and prognosis are rare. We recently reported the role of its interacting partner, MCL1, in oral cancer progression and outcome. Hence, the present study aimed to assess TCTP expression in oral tumorigenesis and its association with patient outcomes alone and in combination with MCL1. METHODS: TCTP expression was assessed by immunohistochemistry and immunoblotting in oral tissues and cells, respectively. Cell viability post siRNA/dihydroartemisinin treatment was analysed by tetrazolium salt assay. Cell survival, invasion and tumorigenic potential post TCTP knockdown were assessed by clonogenic, Matrigel and soft-agar assays, respectively. The association of TCTP with patient outcome was analysed by Kaplan-Meier and Cox regression. RESULTS: TCTP was significantly overexpressed in oral premalignant lesions (p < 0.0001), oral tumours (p < 0.0001) and oral dysplastic and cancer cells versus normal oral mucosa and also in recurrent (p < 0.05) versus non-recurrent oral tumours. Further, elevated TCTP was significantly (p < 0.05) associated with poor recurrence free survival (RFS) and poor overall survival (OS; hazard ratio = 2.29; p < 0.05). Intriguingly, the high co-expression of TCTP and MCL1 further reduced the RFS (p < 0.05) and OS (p < 0.05; hazard-ratio = 3.49; p < 0.05). Additionally, TCTP knockdown decreased survival (p < 0.05), invasion (p < 0.01) and in vitro tumorigenic potential (p < 0.0001). Dihydroartemisinin treatment reduced TCTP levels and viability of oral cancer cells. CONCLUSION: Our studies demonstrate an oncogenic role of TCTP in oral cancer progression and poor outcome. Thus, TCTP may be a potential prognostic marker and therapeutic target in oral cancers.

HPLC-PDA identification and resolution of rufinamide forced degradation impurities: A congregated chemometric expedite optimization coupled with factorials and desirability.

Rufinamide is used presently to treat Lenaux-Gastaut syndrome. A full factorial design and desirability approach was investigated for the optimization of hydrolytic stress via response surface curves (RSCs). The degradation impurities were identified and resolved using reversed-phase high-performance liquid chromatography (RP-HPLC) on the Qualisil® BDS C8 column. Acetonitrile-water (29:71, v/v) was optimized for the mobile phase and used at a flow rate of 1.0 ml/min with detection at a wavelength of 230 nm. Rufinamide showed appreciable susceptibility to hydrolysis under acidic and alkaline stress, and substantial degradation in the neutral condition. It degraded much less under oxidative stress. Exposure towards thermal and photolytic stress conditions indicated appreciable stability. The developed method was subjected to validation as per the recommendations of the International Conference on Harmonization. The proposed method showed no influence from the excipients and the degradation products. As well as good precision and accuracy in determination, the method showed a linear response between 2 and 12 µg ml-1 . The method was extended for determination in a human plasma sample, which resulted in excellent recovery without interference from matrix effects. The combined use of desirability and design for the optimization of acidic and alkaline hydrolytic stress led to simple and rapid analysis.

Elevated USP9X drives early-to-late-stage oral tumorigenesis via stabilisation of anti-apoptotic MCL-1 protein and impacts outcome in oral cancers.

BACKGROUND: Overexpression of anti-apoptotic MCL-1 protein in oral squamous cell carcinoma (OSCC) is linked to disease progression, therapy resistance and poor outcome. Despite its characteristic short half-life owing to ubiquitin-proteasome-dependent degradation, oral tumours frequently show elevated MCL-1 protein expression. Hence, we investigated the role of deubiquitinase USP9X in stabilising MCL-1 protein and its contribution to oral tumorigenesis. METHODS: Expression of MCL-1 and USP9X was assessed by immunoblotting and immunohistochemistry in oral cancer cell lines and tissues. The association between MCL-1 and USP9X was confirmed by coimmunoprecipitation and immunofluorescence. Cell death assessment was performed by MTT, flow cytometry and clonogenic assays. RESULTS: Both USP9X and MCL-1 are significantly elevated in oral premalignant lesions and oral tumours versus normal mucosa. USP9X interacts with and deubiquitinates MCL-1, thereby stabilising it. Pharmacological inhibition of USP9X potently induced cell death in OSCC cells in vitro and in vivo. The elevated expression of USP9X and MCL-1 correlated with poor prognosis in OSCC patients. CONCLUSION: We demonstrate the oncogenic role of USP9X in driving early-to-late stages of oral tumorigenesis via stabilisation of MCL-1, suggesting its potential as a prognostic biomarker and therapeutic target in oral cancers.

Comparison of cleaning effectiveness of single rotary file OneShape and reciprocating F2 Protaper with Protaper Universal sequence: A SEM analysis.

AIM: This in vitro study intend to compare the cleaning effectiveness of Protaper universal sequence with reciprocating F2 Protaper and single rotary file One shape. MATERIALS AND METHOD: 30 extracted human 1st mandibular molars were chosen for the analysis. Three NiTi file systems were used for mechanical preparation, ProTaper full sequence in rotary motion, single F2 Protaper file used in reciprocating motion, and One shape single file used in a circular motion. Irrigation was carried out after each instrument use using 5 ml of 5% NaOCl followed by normal saline. The root canal surface was evaluated at three different areas (coronal, middle and apical thirds) using Scanning Electron Microscopy. Debris and the Smear layer were evaluated. Data were analyzed statistically using the Friedman test and Kruskal-Wallis test (p ≤ 0.05). RESULTS: A statistically significant difference (p ≤ 0.05) was observed in the debris score of the Protaper universal group when the 3 thirds of the root were compared. Intergroup comparisons confirmed a statistically significant difference at the coronal and apical third of the roots when debris scores were evaluated. Intragroup comparison for the smear layer demonstrated a statistically significant difference (p ≤ 0.05) at all the 3 levels of the radicular canal for the 3 groups studied. Intergroup comparisons revealed a statistically significant difference (p ≤ 0.05) in the middle and apical 1/3rd when the smear layer was evaluated. CONCLUSION: The Protaper full sequence group provided better results than Single F2 ProTaper and One shape groups when debris and smear layer removal was investigated.

Concomitant overexpression of Activin A and p63 is associated with poor outcome in oral cancer patients.

BACKGROUND: The present study aims to comprehensively analyze expression of Activin signaling components in oral cancer and to determine the predominant Activin expressed and its influence on prognosis. As our preliminary studies indicated regulation of Activin gene by p63, we also propose to assess its correlation with p63/p53 in oral tumors and its impact on outcome. METHODS: Expression of Activin subunits, receptors, and regulators was assessed by qRT-PCR and Western blotting. Correlation between Activin A and p63/p53 expression was evaluated in oral tumors by immunohistochemistry and their association with clinical outcome was determined by Kaplan-Meier curves and Cox regression. RESULTS: Activin ßA transcripts were upregulated (P = .013) in oral dysplastic and cancer cells compared with normal oral mucosa. Expression of Activin receptors and regulators was also altered. Activin ßA protein was significantly upregulated in oral tumors and adjacent normal tissues compared with normal oral mucosa (P < .0001). Expression of Activin ßA and p63 significantly correlated in oral tumors, correlation being stronger in tumors with high p53 (r = -.394, P = .005). Activin ßA overexpression was associated with advanced tumor stage (P = .021), positive nodes (P = .045), poor recurrence-free survival (P = .013), and overall survival (P = .024), while its concomitant overexpression with p63 was a better predictor of recurrence-free survival (HR = 10.66, CI: 1.41-80.19). CONCLUSIONS: Activin A overexpression is an early event in oral cancer pathogenesis and can independently predict survival. Moreover, in combination with p63 overexpression, it served as a better marker for poor prognosis. Activin A could thus be a promising target for improved outcome in oral cancer patients.

Human Papillomavirus-Related Multiphenotypic Sinonasal Carcinoma with Unique HPV type 52 Association: A Case Report with Review of Literature.

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) is a recently described distinctive clinicopathologic entity defined by association to high risk HPV, localization to sinonasal tract and close histologic resemblance to salivary gland tumors. Lack of awareness of its pathologic features and biology among pathologists and oncologists make this entity susceptible to misdiagnosis and erroneous management. Herein, we illustrate a case of HMSC of the nasal cavity associated with heretofore unreported subtype HPV-52 and discuss the challenges associated with diagnosis and management of this rare tumor. A 48-year-old woman with intermittent epistaxis for 6 months presented with a nasal mass and underwent middle turbinectomy. Histology showed a tumor with features typical of adenoid cystic carcinoma (ACC) in the form of basaloid cells and cribriform architecture. However, careful inspection revealed findings uncommon in ACC; such as surface pagetoid tumor spread, areas of solid sheets of myoepithelial cells accompanied by increased mitotic figures which prompted immunohistochemistry. Multidirectional differentiation into ductal (CK7, AE1/AE3) and myoepithelial (p63, p40, S100, calponin) lineage together with strong and diffuse immunopositivity for p16 distinguished this tumor from ACC. HPV genotyping was positive for high risk HPV subtype HPV52, which confirmed the diagnosis of HMSC. HPV-related multiphenotypic sinonasal carcinoma is an under-recognized unique clinicopathologic entity that needs awareness to avoid mistaking it for commoner salivary gland tumors. Making accurate diagnosis of this newly-described tumor is imperative in order to understand its biology and to develop optimal therapeutic strategies.

Impact of HPV 16/18 infection on clinical outcomes in locally advanced cervical cancers treated with radical radio (chemo) therapy - A prospective observational study.

OBJECTIVE: With an aim to investigate the impact of Human Papilloma Virus (HPV) 16/18 infection on clinical outcomes in locally advanced cervical cancers treated with radical radio (chemo) therapy, we undertook this prospective study. METHODS: Between May 2010 and April 2012, 150 histologically proven cervical cancer patients treated with radio (chemo) therapy were accrued. Cervical biopsies/brushings were collected at pre-treatment, end of treatment and at 3 monthly intervals up to 24months. Quantitative estimation of HPV 16/18 was done using real-time polymerase chain reaction (RT-PCR) and correlated with various clinical end-points. RESULTS: Out of 150 patients accrued, 135 patients were considered for final analysis. Pre-treatment HPV16/18 DNA was detected in 126 (93%) patients, with HPV-16 present in 91%. The mean log (±SD) HPV-16 and HPV-18 viral load at pre-treatment was 4.76 (±2.5) and 0.14 (±2.1) copies/10ng of DNA, respectively. Though significant decline in viral load was observed on follow-ups (p<0.0001); by 9-month follow-up, 89 (66%) patients had persistence of HPV infection. Patients with persistent HPV 16/18 infection had a significantly higher overall and loco-regional relapses [44/89 (49%) and 29/89 (32%)] as compared to HPV clearance by 9months [12/43 (28%) and 5/43 (11%)] with p=0.024 and p=0.02, respectively. Also, persistent HPV infection by 24-month showed a significant impact on loco-regional control (LRC) and recurrence-free survival (RFS). CONCLUSION: In locally advanced cervical cancers treated with radical radio (chemo) therapy, persistent HPV 16/18 infection is significantly high in immediate post-treatment period and correlated with higher loco-regional, overall relapses and was also associated with early relapses.

Human papillomavirus in head and neck cancer in India: Current status and consensus recommendations.

Human papillomavirus (HPV) associated head and neck squamous cell cancers (HNSCC) have become increasingly common in the West, but the same cannot be said about India. These cancers have a different biology and confer a better prognosis, however, its current role in the management of patients in India is not clearly defined. At the 35th Indian Cooperative Oncology Network conference held in September 2016, a panel of radiation, surgical and medical oncologists, pathologists, and basic scientists from across the country having experience in clinical research with respect to HPV in HNSCC reviewed the available literature from India. All the ideas and facts were thereafter collated in this report. Various topics of controversy in dealing with the diagnosis and management of HPV-associated HNSCC have been highlighted in this report in context to the Indian scenario. Furthermore, the prevalence of the same and its association with tobacco and high-risk sexual behavior has been touched on. Conclusively, a set of recommendations has been proposed by the panel to guide the practicing oncologists of the country while dealing with HPV-associated HNSCC.

High expression of survivin and its splice variants survivin ΔEx3 and survivin 2 B in oral cancers.

OBJECTIVES: We have previously reported inactivation of p53 in 46% of Indian patients with oral cancer. Survivin, a p53 target gene and an inhibitor of apoptosis protein (IAP), is overexpressed in several cancers, including oral cancers. Studies assessing the role of survivin and its splice variants in oral cancers are, however, rare. MATERIALS AND METHODS: The expression of 6 survivin isoforms in 4 oral cancer cell lines (AW8507, AW13516, UPCI-SCC040, UPCI-SCC029 B), a dysplastic oral cell line (DOK), 75 paired oral tumor and adjacent normal tissues, and 12 normal oral tissue samples from healthy individuals was analyzed by real-time PCR. The expression was correlated with clinicopathologic parameters, which included age, sex, tumor-node-metastasis (TNM) staging, tobacco and/or alcohol consumption, site, and differentiation status of tumor. RESULTS: This is the first study to find overexpression of the 6 characterized survivin isoforms in oral cancers compared with normal tissues (P < .05). Additionally, a significant (P < .05) correlation among the fold changes of all 6 survivin isoforms was observed. Survivin wild type (wt) was the predominantly expressed isoform in oral cell lines and tumor tissues versus normal tissues (P < .05). Among the minor isoforms, survivin ΔEx3 and survivin 2 B were dominantly expressed, whereas survivin 2 α and survivin 3 α overexpression was found for the first time. Further high survivin 3 B expression exhibited a significant association (P < .05) with poorly differentiated tumors. Interestingly the combined expression of the antiapoptotic survivin isoforms, survivin wt, survivin ΔEx3, and survivin 3 B, exhibited a significant association with TNM staging of the tumor. CONCLUSIONS: Our studies thus indicate that oral cancers overexpress the antiapoptotic survivin variants, which exhibit an association with advanced tumor stage, implying a role for these variants in oral tumorigenesis.

A simple cost-effective modification improves the quality of immunocytochemical staining in cervical scrape samples characterized by presence of excess mucus.

Immunocytochemistry (ICC) is a very important tool in a diverse range of biomedical research as well as in diagnostic cytopathology. Smears prepared from cervical scrapes contain a large amount of overlying mucus that interferes with the standard immunocytochemical staining protocol. A modified ICC protocol is described, which involves pretreatment of these smears with 1 mg/ml solution of Ambroxol hydrochloride in methanol for 1 hour. Source of Ambroxol hydrochloride was a 30 mg Mucolite™ tablet, at a cost of 1.70 rupees (∼3·5 US cents) per tablet. This mucolytic solution effectively clears the mucus, facilitating the accessibility of the antibody to the antigenic determinants. This pretreatment resulted in the increased percentage of positively stained cells as well as staining intensity, leading to improved overall ICC staining and score. This is a novel modification that can be cost-effectively applied in ICC staining protocols for cytology samples characterized by the presence of excess mucus.

Bcl-xL protein: predictor of complete tumor response in patients with oral cancer treated with curative radiotherapy.

BACKGROUND: We earlier observed altered expression of p53 and Bcl-xL in oral cancer cell lines/tissues and wanted to evaluate these proteins for prediction of radiotherapy response and outcome. METHODS: Thirty-nine paraffin-embedded, pretreatment oral cancer biopsies were analyzed for protein expression using immunohistochemistry and correlated with tumor response to radiotherapy and disease-free survival (DFS). RESULT: High p53 (p = .040) was observed in female versus male patients. Increased p53 intensity (p = .063) was observed in tobacco habitués (chewers ± smokers) versus patients with no habits. In univariate analysis, nodal positivity (p = .044) and favorable/complete tumor response (p = .002) exhibited a significant correlation with DFS, whereas tumor response emerged as an independent predictor of DFS in multivariate analysis. Significantly high Bcl-xL (p = .048) was observed in the unfavorable versus favorable responders. CONCLUSION: Our study suggests that Bcl-xL expression along with clinical parameters may be useful for identifying patients with oral cancer likely to draw maximum benefit from curative radiotherapy.

PCNA and anti-apoptotic Mcl-1 proteins predict disease-free survival in oral cancer patients treated with definitive radiotherapy.

At our laboratory, we recently observed cell cycle and apoptosis-related proteins Myeloid Cell Leukemia-1 (Mcl-1) and Proliferating Cell Nuclear Antigen (PCNA) to be altered in oral tumours/cell lines. The present study aimed to evaluate the above proteins for predicting response and outcome in oral cancer patients treated with definitive radiotherapy. Pre-treatment oral cancer biopsy samples from 39 patients were examined for Mcl-1 and PCNA proteins using immunohistochemistry and correlated with clinico-pathological variables using disease-free survival (DFS) as the primary endpoint. We observed high expression of Mcl-1 in older versus younger patients (p=0.013) and in tobacco chewers+/-alcohol versus smokers+/-alcohol (p=0.037); and PCNA in node-positive versus node-negative tumours (p=0.037). On univariate analysis, high PCNA (p=0.007), Mcl-1 (p=0.050), node positivity (p=0.040) and co-expression of PCNA and Mcl-1 (p=0.008), had a significant impact on DFS. On multivariate analysis, low PCNA/Mcl-1 (p=0.006) co-expressing tumours were associated with improved DFS. Thus our study suggests that in patients undergoing primary radiotherapy, PCNA could be of potential predictive value to identify patients with risk of nodal metastases and in combination with Mcl-1 may have potential prognostic value to differentiate patients with poor DFS. These markers may be used for future trial patients requiring radiotherapy for their treatment.

Expression of bcl-2 and bax in chewing tobacco-induced oral cancers and oral lesions from India.

Deregulation of oncogenes and tumor suppressor genes involved in apoptosis has been associated with tumor development and progression. To investigate the involvement of apoptosis regulating proteins in oral cancer in Indian patients, primarily associated with chewing tobacco habits, immunohistochemical expression of bcl-2 and bax was examined in 63 oral squamous cell carcinomas, and 31 putative premalignant lesions. Our studies revealed overexpression of tumor specific cytoplasmic bcl-2 in 56% and bax in 43% oral cancers. The oral cancers in the Indian patients are preceded by premalignant oral lesions; hence oral lesions were examined for bcl-2 and bax expression. We observed aberrant expression of bcl-2 in 16% oral lesions comprising leukoplakias and SMF and bax in 55% oral lesions. We have already reported, p53 expression in these oral cancers and lesions. It was noteworthy that 30% oral cancers demonstrated a p53+bcl2+ pattern, and 14% samples exhibited p53+bcl2+bax+ pattern. However, none of the oral lesions showed concurrent deregulation of p53 and bcl-2 or all the three genes. Interestingly 45% oral lesions were p53-bax+ as compared to 18% oral cancers; while 39% oral lesions were bcl2-bax+ as compared to 14% oral cancers, indicating overexpression of bax in oral lesions, in the absence of p53 and bcl-2 proteins. Significant correlation was observed between positive nodal status and bcl2+ (p=0.047) and p53+bcl-2+ (p=0.01) in oral cancers. Kaplan Meier survival analysis showed significantly (p=0.059) higher survival in patients with p53- oral tumors than with p53+ tumors. Our studies thus indicate frequent overexpression of apoptosis regulators bcl-2, bax and p53 proteins in oral cancers, and a subset of oral lesions, representing early events in oral car-cinogenesis. The aberrant bcl-2 expression and loss of p53 function observed, may play an important role in the tumorigenesis of oral cancers by allowing escape from apoptosis and enabling additional genetic alterations to accrue.