Thearubigins/Polymeric black tea polyphenols (PBPs) do not prevent benzo[a]pyrene (B[a]P) induced lung tumors in A/J mice.

OBJECTIVES: Previously we have demonstrated the chemopreventive effect of Thearubigins/Polymeric Black-tea Polyphenols (PBPs) upon pre-treatment to a combination of carcinogens, that is, Benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) which are present in Tobacco smoke (TS). However, the chemopreventive effect in response to B[a]P as a single carcinogen remains unexplored. B[a]P is a universal carcinogen and an important constituent of particulate matter 2.5 (PM2.5) found in the environment and in TS. METHODS: We investigated the pre-treatment of Thearubigins/PBPs as a chemopreventive agent at three doses (1.5, 5, 10%) against B[a]P-induced lung carcinogenesis at early & late time points. We also investigated the effect of PBPs at early time points to understand molecular changes by employing western blotting in xenobiotic metabolism pathways, DNA damage, inflammation, apoptosis, and proliferation as they are modulated in response to carcinogens. We used 6-8 weeks male A/J mice for tumorigenicty and western blotting to probe the molecular biomarkers. RESULTS: We report no decrease in tumor incidence and multiplicity upon pre and concurrent treatment of Thearubigins/PBPs. Further, we also report no changes in molecular markers at early time points, in agreement with former observations. CONCLUSION: Our results suggest that chemopreventive agents need to be tested with different combinations of carcinogens and regimens to fully understand the complex interplay between carcinogenesis and the efficacy of chemopreventive agents. Studies like these will provide meaningful data before initiating large-scale clinical trials.

Role of DNA methylation-based mitotic ageing indices in oral cancer development and recurrence.

OBJECTIVE: DNA methylation data can be used to derive mitotic indices from complex tissues. Here, we assessed if the DNA methylation-derived mitotic ageing indices are associated with oral squamous cell carcinoma (OSCC) development and recurrence-free survival (RFS). METHODS: DNA methylation-based mitotic indices (MitoticAge, TNSC and hypoSC) were derived using algorithms "MitoticAge" and "epiTOC2" for the discovery [non-malignant (n = 22), premalignant (n = 22) and OSCC (n = 68) tissues] and validation datasets (GSE87053, GSE136704 and TCGA-HNSCC). Differences in mitotic indices between non-malignant, premalignant and OSCC tissues were assessed. Finally, the association between estimated mitotic indices and RFS was evaluated in OSCCs. RESULTS: In the discovery and validation datasets, increased mitotic ageing was observed in OSCC compared to non-malignant and premalignant oral tissues. HPV-positive HNSCCs had higher mitotic index TNSC. Mitotic age index hypoSC was associated with RFS in OSCC (p = 0.011, HR 2.61, 95% CI 1.24-5.48). CONCLUSIONS: DNA methylation-derived mitotic indices are associated with OSCC development and RFS. Thus, DNA methylation-derived mitotic indices may be a valuable research tool to reliably estimate the cumulative number of stem cell divisions in malignant and non-malignant oral tissues. Future research utilizing mitotic indices for predicting clinical outcomes in OSCC is warranted.

Genome-wide DNA methylation profiling of HPV-negative leukoplakia and gingivobuccal complex cancers.

BACKGROUND: Gingivobuccal complex oral squamous cell carcinoma (GBC-OSCC) is an aggressive malignancy with high mortality often preceded by premalignant lesions, including leukoplakia. Previous studies have reported genomic drivers in OSCC, but much remains to be elucidated about DNA methylation patterns across different stages of oral carcinogenesis. RESULTS: There is a serious lack of biomarkers and clinical application of biomarkers for early detection and prognosis of gingivobuccal complex cancers. Hence, in search of novel biomarkers, we measured genome-wide DNA methylation in 22 normal oral tissues, 22 leukoplakia, and 74 GBC-OSCC tissue samples. Both leukoplakia and GBC-OSCC had distinct methylation profiles as compared to normal oral tissue samples. Aberrant DNA methylation increases during the different stages of oral carcinogenesis, from premalignant lesions to carcinoma. We identified 846 and 5111 differentially methylated promoters in leukoplakia and GBC-OSCC, respectively, with a sizable fraction shared between the two sets. Further, we identified potential biomarkers from integrative analysis in gingivobuccal complex cancers and validated them in an independent cohort. Integration of genome, epigenome, and transcriptome data revealed candidate genes with gene expression synergistically regulated by copy number and DNA methylation changes. Regularised Cox regression identified 32 genes associated with patient survival. In an independent set of samples, we validated eight genes (FAT1, GLDC, HOXB13, CST7, CYB5A, MLLT11, GHR, LY75) from the integrative analysis and 30 genes from previously published reports. Bisulfite pyrosequencing validated GLDC (P = 0.036), HOXB13 (P < 0.0001) promoter hypermethylation, and FAT1 (P < 0.0001) hypomethylation in GBC-OSCC compared to normal controls. CONCLUSIONS: Our findings identified methylation signatures associated with leukoplakia and gingivobuccal complex cancers. The integrative analysis in GBC-OSCC identified putative biomarkers that enhance existing knowledge of oral carcinogenesis and may potentially help in risk stratification and prognosis of GBC-OSCC.

Prevention of carcinogen-induced oral cancers by polymeric black tea polyphenols via modulation of EGFR-Akt-mTOR pathway.

The overexpression of Epidermal Growth Factor Receptor (EGFR) and dysregulation of its downstream effector pathways are important molecular hallmarks of oral cancers. Present study investigates the chemopreventive potential of polymeric black tea polyphenols (PBPs)/thearubigins (TRs) in the hamster model of oral carcinogenesis as well as determine the effect of PBPs on EGFR and the molecular players in the EGFR pathway. In dose-dependent manner, pre and concurrent treatment with PBPs (1.5%, 5%, 10%) decreased the number and volume of macroscopic tumors as well as the number and area of microscopic lesions. Interestingly, at 10% dose of PBPs, no macroscopic or microscopic tumors were observed. We observed PBPs mediated dose-dependent decrease in oxidative DNA damage (8OHdG); inflammation (COX-2); proliferation (PCNA, Cyclin D1); expression of EGFR, and its downstream signaling kinases (pAkt, Akt, and mTOR); hypoxia (HIF1α) and angiogenesis (VEGF). There was also a PBPs mediated dose-dependent increase in apoptosis (Bax). Thus, our data clearly indicate that the observed chemopreventive potential of PBPs was due to modulation in the EGFR pathway associated with cell proliferation, hypoxia, and angiogenesis. Taken together, our results demonstrate preclinical chemopreventive efficacy of PBPs and give an insight into its mechanistic role in the chemoprevention of experimental oral cancer.

Prognostic and predictive roles of cancer stem cell markers in head and neck squamous cell carcinoma patients receiving chemoradiotherapy with or without nimotuzumab.

BACKGROUND: Anti-EGFR-based therapies have limited success in HNSCC patients. Predictive biomarkers are needed to identify the patients most likely to benefit from these therapies. Here, we present predictive and prognostic associations of different cancer stem cell markers in HPV-negative locally advanced (LA) HNSCC patients. METHODS: Pretreatment tumour tissues of 404 HPV-negative LA-HNSCCs patients, a subset of-phase 3-randomised study comparing cisplatin-radiation(CRT) and nimotuzumab plus cisplatin-radiation(NCRT) were examined. The expression levels of CD44, CD44v6, CD98hc, ALDH1A1, SOX2 and OCT4A were evaluated using immunohistochemistry. Progression-free survival(PFS), loco-regional control(LRC),- and overall survival(OS) were estimated by Kaplan-Meier method. Hazard ratios were estimated by Cox proportional hazard models. RESULTS: NCRT showed significantly improved OS with low membrane expression of CD44 compared to CRT [HR (95% CI) = 0.63 (0.46-0.88)]. Patients with low CD44v6 also showed better outcomes with NCRT [LRC: HR (95% CI) = 0.25 (0.10-0.62); OS: HR (95% CI) = 0.38 (0.19-0.74)]. No similar benefit with NCRT observed in patients with high CD44 or CD44v6 expression. Bootstrap resampling confirmed the predictive effect of CD44 (Interaction P = 0.015) and CD44v6 (Interaction P = 0.041) for OS. Multivariable Cox analysis revealed an independent negative prognostic role of CD98hc membrane expression for LRC [HR (95% CI) = 0.63(0.39-1.0)] and OS[HR (95% CI) = 0.62 (0.40-0.95)]. CONCLUSIONS: CD44 and CD44v6 are potential predictive biomarkers for NCRT response. CD98hc emerged as an independent negative prognostic biomarker. CLINICAL TRIAL REGISTRATION: Registered with the Clinical Trial Registry of India (Trial registration identifier-CTRI/2014/09/004980).

Correlation between imaging and tissue biomarkers of hypoxia in squamous cell cancer of the head and neck.

The aim of this study was to correlate endogenous tissue biomarkers of hypoxia with quantitative imaging parameters derived from 18F-fluoro-misonidazole (F-MISO) and 18F-fluoro-deoxy-glucose (FDG) positron emission tomography/computed tomography (PET/CT) and clinical outcomes in locoregionally advanced head and neck squamous cell carcinoma (HNSCC). Tumor-tissue blocks of HNSCC patients with pretreatment F-MISO-PET/CT and FDG-PET/CT were de-archived for expression of hypoxia-inducible factor-1 alpha (HIF-1α) subunit, carbonic anhydrase-IX (CA-IX), and glucose transporter subunit-1 (GLUT-1) using immunohistochemistry (IHC). The intensity of staining was graded and correlated with quantitative imaging parameters and with disease-related outcomes. Tissue blocks were analyzed for 14 of 20 patients. On IHC, median H-scores for HIF-1α, CA-IX, and GLUT-1 were 130, 0, and 95, respectively. No significant correlation of tissue biomarkers of hypoxia with quantitative imaging parameters was found. However, borderline significant correlation was seen for H-scores of CA-IX with hypoxic tumor volume (HTV) (r = 0.873, P = 0.054) and fractional hypoxic volume (r = 0.824, P = 0.086) derived from F-MISO-PET/CT. At a median follow-up of 43 months, 5-year Kaplan-Meier estimates of locoregional control, disease-free survival, and overall survival were 53%, 43%, and 40%, respectively. Increased expression of HIF-1α or GLUT-1 (dichotomized by median H-scores) was not individually associated with disease-related outcomes. However, a combination of high HTV (>4.89cc) with above median H-scores of either HIF-1α (>130) and/or GLUT-1 (>95) was associated with worse clinical outcomes. None of the three patients with such "adverse hypoxic profile" were long-term survivors. There is no significant correlation of endogenous tissue biomarkers of hypoxia (HIF-1α, CA-IX, and GLUT-1) with quantitative imaging parameters (on F-MISO-PET/CT and FDG-PET/CT) or long-term outcomes in HNSCC. However, a combination of both can identify a subgroup of patients with adverse outcomes.

Correlation of transcriptionally active human papillomavirus status with the clinical and molecular profiles of head and neck squamous cell carcinomas.

BACKGROUND: To examine the molecular profiles of human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinomas (HNSCCs), expression of epidermal growth factor receptor (EGFR), phospho-EGFR dimers, hypoxia markers, and cancer stem cell markers were evaluated. METHODS: HPV-status was confirmed using RNA-ISH. Immunohistochemical data of biomarker expression levels were analyzed using the Mann-Whitney U test. The clinical outcomes and biomarker expression in the HPV-positive (n = 25), matched HPV-negative (n = 49), and p16-positive/HPV-negative (n = 20) subgroups were comparatively analyzed. RESULTS: HPV was detected in 25 (5.8%) cases and was significantly associated with favorable outcomes. HPV-positive tumors exhibited lower membrane expression of EGFR, pEGFRY1068, pEGFRY1173, CD44, CD44v6, and CD98hc than HPV-negative and p16-positive tumors. The expression of HIF1α, CA9, ALDH1A1, and SOX2 was not significantly associated with HPV-status. The clinical outcomes and biomarker expression levels were similar between the HPV-negative and p16-positive HNSCC. CONCLUSION: HPV-positive HNSCC exhibited distinct molecular profile compared to HPV-negative and p16-positive HNSCC. The clinical and molecular profiles were similar between p16-positive and HPV-negative subgroups.

Prognostic and predictive significance of nuclear HIF1α expression in locally advanced HNSCC patients treated with chemoradiation with or without nimotuzumab.

BACKGROUND: Anti-EGFR-based therapies have limited success in HNSCC patients. Predictive biomarkers are greatly needed to identify the patients likely to be benefited from these targeted therapies. Here, we present the prognostic and predictive association of biomarkers in HPV-negative locally advanced (LA) HNSCC patients. METHODS: Treatment-naive tumour tissue samples of 404 patients, a subset of randomised Phase 3 trial comparing cisplatin radiation (CRT) versus nimotuzumab plus cisplatin radiation (NCRT) were analysed to evaluate the expression of HIF1α, EGFR and pEGFR by immunohistochemistry and EGFR gene copy change by FISH. Progression-free survival (PFS), locoregional control (LRC) and overall survival (OS) were estimated by Kaplan-Meier method. Hazard ratios were estimated by Cox proportional hazard models. RESULTS: Baseline characteristics of the patients were balanced between two treatment groups (CRT vs NCRT) and were representative of the trial cohort. The median follow-up was of 39.13 months. Low HIF1α was associated with better PFS [HR (95% CI) = 0.62 (0.42-0.93)], LRC [HR (95% CI) = 0.56 (0.37-0.86)] and OS [HR (95% CI) = 0.63 (0.43-0.93)] in the CRT group. Multivariable analysis revealed HIF1α as an independent negative prognostic biomarker. For patients with high HIF1α, NCRT significantly improved the outcomes [PFS:HR (95% CI) = 0.55 (0.37-0.82), LRC:HR (95% CI) = 0.55 (0.36-0.85) and OS:HR (95% CI) = 0.54 (0.36-0.81)] compared to CRT. While in patients with low HIF1α, no difference in the clinical outcomes was observed between treatments. Interaction test suggested a predictive value of HIF1α for OS (P = 0.008). CONCLUSIONS: High HIF1α expression is a predictor of poor clinical response to CRT in HPV-negative LA-HNSCC patients. These patients with high HIF1α significantly benefited with the addition of nimotuzumab to CRT. CLINICAL TRIAL REGISTRATION: Registered with the Clinical Trial Registry of India (Trial registration identifier-CTRI/2014/09/004980).

Nimotuzumab-cisplatin-radiation versus cisplatin-radiation in HPV negative oropharyngeal cancer.

BACKGROUND: Addition of nimotuzumab to weekly cisplatin and radiation improves outcomes in head and neck cancer. HPV negative oropharyngeal cancer has unsatisfactory treatment outcomes and is a candidate for escalation of treatment. We wanted to determine whether the addition of nimotuzumab to cisplatin-radiation could improve outcomes in these poor-risk tumors. METHODS: This was a subgroup analysis of a phase 3 randomized study. In this study, locally advanced head and neck cancer patients undergoing definitive chemoradiation were randomly allocated to weekly cisplatin (30 mg/m2 IV)- radiation (66-70 Gy) {CRT arm} or nimotuzumab (200 mg weekly) -weekly cisplatin (30 mg/m2)-radiation (66-70 Gy) {NCRT arm}. The data of HPV negative oropharyngeal cancer was extracted from the database of this study for the analysis. HPV testing was done with p16 immunohistochemistry (IHC) staining and reported according to the CAP criteria. The outcomes assessed were progression-free survival (PFS), disease-free survival (DFS), locoregional control, and overall survival (OS). Interaction test was performed between the study arms and HPV status prior to doing any HPV specific analysis for each of the studied outcomes. Kaplan Meier estimates for 2 year OS with 95%CI was calculated. The hazard ratio was obtained using COX regression analysis. RESULTS: We had 187 HPV negative oropharyngeal cancers, 91 in the CRT arm and 96 in NCRT arm. The interaction test was significant for PFS (p = 0.000), locoregional control (p = 0.007) and overall survival (p = 0.002) but not for DFS (p = 0.072). The 2- year PFS was 31.5% (95%CI 21.5-42) in CRT arm versus 57.2% (95%CI 45.8-67.1) in NCRT arm (HR -0.54; 95%CI 0.36-0.79, p = 0.002). The 2-year LRC was 41.4% (95%CI 29.8-52.6) in the CRT arm versus in 60.4% (95%CI 48.7-70.2) in the NCRT arm (HR -0.61; 95%CI 0.4-0.94, p = 0.024). The addition of nimotuzumab also lead to an improvement in 2-year OS from 39.0% (95%CI 28.4-49.6) to 57.6% (95%CI 46.3-67.4) (HR-0.63, 95%CI 0.43-0.92, p = 0.018). CONCLUSIONS: The addition of nimotuzumab to weekly cisplatin-radiation improves outcomes inclusive of OS in HPV negative oropharyngeal cancers.

Phase I/II Study of Palliative Triple Metronomic Chemotherapy in Platinum-Refractory/Early-Failure Oral Cancer.

PURPOSE: Platinum-resistant oral cancer has a dismal outcome with limited treatment options. We conducted a phase I/II study to identify the optimal biologic dose (OBD) of methotrexate when given along with erlotinib and celecoxib and to assess the efficacy of this three-drug regimen in advanced oral cancer. METHODS: Patients with platinum-resistant or early-failure squamous cell carcinoma of the oral cavity were eligible for this study. They were orally administered erlotinib 150 mg once per day, celecoxib 200 mg twice per day, and methotrexate per week. The primary end point of phase I was to determine the OBD of methotrexate, and that of phase II was to determine the 3-month progression-free survival. The OBD of methotrexate was determined on the basis of the clinical benefit rate at 2 months and circulating endothelial cell level at day 8, using a de-escalation model. Pharmacokinetic evaluation was performed during phase I. Phase II consisted of an expansion cohort of 76 patients. RESULTS: Fifteen patients were recruited in phase I, and 9 mg/m2 methotrexate was identified as the OBD. A total of 91 patients were recruited, and the median follow-up was 6.8 months (range, 0 to 16.8 months). The 3-month progression-free survival rate was 71.1% (95% CI, 60.5% to 79.3%), the 6-month overall survival rate was 61.2% (95% CI, 49.2% to 67.8%), and the response rate was 42.9% (95% CI, 33.2% to 53.1%; n = 39). The mean Functional Assessment of Cancer Therapy-Head and Neck Trial Outcome Index score at day 8 was improved by 6.1 units (standard deviation, 13.6 units) and was maintained around this magnitude (P = .001). CONCLUSION: Triple oral metronomic chemotherapy with erlotinib, methotrexate, and celecoxib is efficacious in platinum-refractory oral cavity cancers and represents a new therapeutic option in patients with poor prognosis.

A randomized phase 3 trial comparing nimotuzumab plus cisplatin chemoradiotherapy versus cisplatin chemoradiotherapy alone in locally advanced head and neck cancer.

BACKGROUND: Because the addition of nimotuzumab to chemoradiation in patients with locally advanced head and neck cancer improved outcomes in a phase 2 study, the authors conducted a phase 3 study to confirm these findings. METHODS: This open-label, investigator-initiated, phase 3, randomized trial was conducted from 2012 to 2018. Adult patients with locally advanced head and neck cancer who were fit for radical chemoradiation were randomized 1:1 to receive either radical radiotherapy (66-70 grays) with concurrent weekly cisplatin (30 mg/m2 ) (CRT) or the same schedule of CRT with weekly nimotuzumab (200 mg) (NCRT).The primary endpoint was progression-free survival (PFS); key secondary endpoints were disease-free survival (DFS), duration of locoregional control (LRC), and overall survival (OS). An intent-to-treat analysis also was performed. RESULTS: In total, 536 patients were allocated equally to both treatment arms. The median follow-up was 39.13 months. The addition of nimotuzumab improved PFS (hazard ratio [HR], 0.69; 95% CI, 0.53-0.89; P = .004), LRC (HR, 0.67; 95% CI, 0.50-0.89; P = .006), and DFS (HR, 0.71; 95% CI, 0.55-0.92; P = .008) and had a trend toward improved OS (HR, 0.84; 95% CI, 0.65-1.08; P = .163). Grade 3 through 5 adverse events were similar between the 2 arms, except for a higher incidence of mucositis in the NCRT arm (66.7% vs 55.8%; P = .01). CONCLUSIONS: The addition of nimotuzumab to concurrent weekly CRT improves PFS, LRC, and DFS. This combination provides a novel alternative therapeutic option to a 3-weekly schedule of 100 mg/m2 cisplatin in patients with locally advanced head and neck cancer who are treated with radical-intent CRT.

Dose-Related Modulatory Effects of Polymeric Black Tea Polyphenols (PBPs) on Initiation and Promotion Events in B(a)P and NNK-Induced Lung Carcinogenesis.

Our understanding of dose-related effects of polymeric black tea polyphenols (PBPs), the most abundant polyphenols in black tea, is limited. In the present study, the effect of various doses of black tea (0.75, 1.5, and 3%)-derived PBP-rich extract on biochemical parameters and lung carcinogenicity in A/J mice was investigated. Pretreatment with PBPs showed the dose-related decrease in B(a)P-induced expression and activity of CYP1A1 in the liver while CYP1A2 expression and activity in the lung. Dose-dependent significant increase in PBP-mediated over-expression and activity of GSTs (alpha in the liver while pi in the lung) were observed in polyphenol-treated groups. Significant dose-related decrease in number and intensity of BPDE-DNA adducts were observed in liver and lung. Black tea (1.5%, 3%)-derived PBPs showed dose-mediated decrease in lung tumor incidence and multiplicity which was further correlated with different molecular markers like cell proliferation and apoptosis in B(a)P and NNK model. In conclusion, dose-dependent chemopreventive effects of PBPs, both anti-initiating (induction of phase II and inhibition of carcinogen-induced phase-I enzymes leading to decrease in BPDE-DNA adducts) and anti-promoting (decreased cell proliferation and increased apoptosis lowering incidence and/or multiplicity of lung lesions), were observed in A/J mice without significant toxicity.

Detection of HPV E6/E7 mRNA in Clinical Samples Using RNA In Situ Hybridization.

Detection of human papilloma virus (HPV) in tissue specimens has been a clinical challenge since last 2 decades; however, screening for presence of E6/E7 transcripts is regarded as the gold standard, and it verifies the active HPV infection. Here, we describe "RNAscope® assay" a novel RNA in situ hybridization (ISH) technology; which detects E6/E7 mRNA of seven high risk HPV subtypes (HPV 16, 18, 31, 33, 35, 52, and 58) in formalin fixed paraffin embedded (FFPE) tissue samples.

Recurring Amplification at 11q22.1-q22.2 Locus Plays an Important Role in Lymph Node Metastasis and Radioresistance in OSCC.

A key feature in the pathogenesis of OSCC is genetic instability, which results in altered expression of genes located in amplified/deleted chromosomal regions. In a previous study we have shown that the amplification of the 11q22.1-q22.2 region, encoding cIAP1 and cIAP2, is associated with lymph node metastasis and poor clinical outcome in OSCC. Here, we validate the aCGH results by nuc ish and detect a weak amplification at the 11q22.1-q22.2 locus in 37% of the 182 samples tested. We find positive correlation of 11q22.1-q22.2 amplification with lymph node metastasis, reduced survival, and increased cancer recurrence, and we observe that patients with 11q22.1-q22.2 amplification fail to respond to radiotherapy. We confirm the concurrent overexpression of cIAP1 and cIAP2 and observe differential subcellular localization of the two proteins in OSCC. To ascertain the roles of cIAP1/cIAP2 in lymph node metastasis and radioresistance, we use an in vitro pre-clinical model and confirm the role of cIAP1 in invasion and the role of cIAP2 in invasion and migration. Studies of other tumor types in which cIAP1 is overexpressed suggest that multi-regimen treatments including SMAC mimetics may be effective. Thus, the evaluation of 11q22.1-q22.2 amplifications in OSCC patients may help choose the most effective treatment.

Raman exfoliative cytology for oral precancer diagnosis.

Oral premalignant lesions (OPLs) such as leukoplakia, erythroplakia, and oral submucous fibrosis, often precede oral cancer. Screening and management of these premalignant conditions can improve prognosis. Raman spectroscopy has previously demonstrated potential in the diagnosis of oral premalignant conditions (in vivo), detected viral infection, and identified cancer in both oral and cervical exfoliated cells (ex vivo). The potential of Raman exfoliative cytology (REC) in identifying premalignant conditions was investigated. Oral exfoliated samples were collected from healthy volunteers (n=20), healthy volunteers with tobacco habits (n=20), and oral premalignant conditions (n=27, OPL) using Cytobrush. Spectra were acquired using Raman microprobe. Spectral acquisition parameters were: λex: 785 nm, laser power: 40 mW, acquisition time: 15 s, and average: 3. Postspectral acquisition, cell pellet was subjected to Pap staining. Multivariate analysis was carried out using principal component analysis and principal component-linear discriminant analysis using both spectra- and patient-wise approaches in three- and two-group models. OPLs could be identified with ∼77% (spectra-wise) and ∼70% (patient-wise) sensitivity in the three-group model while with 86% (spectra-wise) and 83% (patient-wise) in the two-group model. Use of histopathologically confirmed premalignant cases and better sampling devices may help in development of improved standard models and also enhance the sensitivity of the method. Future longitudinal studies can help validate potential of REC in screening and monitoring high-risk populations and prognosis prediction of premalignant lesions.

Chromosomal Alterations and Gene Expression Changes Associated with the Progression of Leukoplakia to Advanced Gingivobuccal Cancer.

We present an integrative genome-wide analysis that can be used to predict the risk of progression from leukoplakia to oral squamous cell carcinoma (OSCC) arising in the gingivobuccal complex (GBC). We find that the genomic and transcriptomic profiles of leukoplakia resemble those observed in later stages of OSCC and that several changes are associated with this progression, including amplification of 8q24.3, deletion of 8p23.2, and dysregulation of DERL3, EIF5A2, ECT2, HOXC9, HOXC13, MAL, MFAP5 and NELL2. Comparing copy number profiles of primary tumors with and without lymph-node metastasis, we identify alterations associated with metastasis, including amplifications of 3p26.3, 8q24.21, 11q22.1, 11q22.3 and deletion of 8p23.2. Integrative analysis reveals several biomarkers that have never or rarely been reported in previous OSCC studies, including amplifications of 1p36.33 (attributable to MXRA8), 3q26.31 (EIF5A2), 9p24.1 (CD274), and 12q13.2 (HOXC9 and HOXC13). Additionally, we find that amplifications of 1p36.33 and 11q22.1 are strongly correlated with poor clinical outcome. Overall, our findings delineate genomic changes that can be used in treatment management for patients with potentially malignant leukoplakia and OSCC patients with higher risk of lymph-node metastasis.

Polymeric black tea polyphenols (PBPs) inhibit benzo(a)pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone-induced lung carcinogenesis potentially through down-regulation of p38 and Akt phosphorylation in A/J mice.

The aim of our study was to evaluate chemopreventive efficacy and possible mechanism of most abundant polyphenolic fraction in black tea, polymeric black tea polyphenols (PBPs), in experimental lung carcinogenesis model. Effect of 1.5% black tea derived PBPs on benzo(a)pyrene [B(a)P] and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced lung lesions were studied over 28 wks. Chemopreventive efficacy was studied using decrease in tumor incidence and/or multiplicity and/or delay in the latency period in A/J mice. Histopathological analysis of lung was carried out post-carcinogen treatment weeks to analyze the microscopic lung lesions. Inflammation, cell proliferation, and apoptosis markers along with signaling kinases like p38, Akt, and their phosphorylated forms were studied using immunoblotting and immunohistochemistry at 4th, 10th, and 18th wk post-carcinogen treatment. Administration of PBPs throughout the treatment period significantly decreased the multiplicity of surface tumors as well as microscopic lung lesions, including adenomas. Although tumor incidence and latency period remains unaffected, histopathological evaluation of lung at 6, 10, and 18 wks post- carcinogen treatment period showed decrease in tumor multiplicity which was also correlated with different molecular markers. Anti- inflammatory action of PBPs was demonstrated by reduced Cox-2 expression. PBPs down-regulated the B(a)P and NNK-induced cell proliferation (diminished PCNA expression, proliferation index, and Bcl-2 expression) and enhanced apoptosis (increased Bax expression and apoptotic index) potentially through phosphorylation of p38 and Akt. PBPs, most abundant polyphenolic component in the black tea, have chemopreventive effect through inhibition of inflammation, cellular proliferation, and induction of apoptosis possibly via modulation of signaling kinases. © 2016 Wiley Periodicals, Inc.

Low prevalence of transcriptionally active human papilloma virus in Indian patients with HNSCC and leukoplakia.

OBJECTIVES: In the present study, we comprehensively analyzed the prevalence of transcriptionally active human papilloma virus (HPV) in tissue samples of Indian patients with leukoplakia, predominantly hyperplastic lesions and head and neck squamous cell carcinoma (HNSCC). In addition, saliva samples from patients with HNSCC were screened for HPV detection. STUDY DESIGN: P16 overexpression was analyzed by immunohistochemistry. Tissue samples of leukoplakia (n = 121) and HNSCC (n = 427) and saliva from patients with HNSCC (n = 215) were tested for HPV using nested polymerase chain reaction. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in situ hybridization. RESULTS: P16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, in saliva or tissue. E6/E7 mRNA positivity was observed in 8 HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx, with very little concordance between p16 overexpression and HPV positivity. All patients with HPV-positive saliva samples had transcriptionally active HPV present in their tumors. CONCLUSION: The presence of HPV DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV-associated tumors.

Understanding the molecular mechanisms of cancer prevention by dietary phytochemicals: From experimental models to clinical trials.

Chemoprevention is one of the cancer prevention approaches wherein natural/synthetic agent(s) are prescribed with the aim to delay or disrupt multiple pathways and processes involved at multiple steps, i.e., initiation, promotion, and progression of cancer. Amongst environmental chemopreventive compounds, diet/beverage-derived components are under evaluation, because of their long history of exposure to humans, high tolerability, low toxicity, and reported biological activities. This compilation briefly covers and compares the available evidence on chemopreventive efficacy and probable mechanism of chemoprevention by selected dietary phytochemicals (capsaicin, curcumin, diallyl sulphide, genistein, green/black tea polyphenols, indoles, lycopene, phenethyl isocyanate, resveratrol, retinoids and tocopherols) in experimental systems and clinical trials. All the dietary phytochemicals covered in this review have demonstrated chemopreventive efficacy against spontaneous or carcinogen-induced experimental tumors and/or associated biomarkers and processes in rodents at several organ sites. The observed anti-initiating, anti-promoting and anti-progression activity of dietary phytochemicals in carcinogen-induced experimental models involve phytochemical-mediated redox changes, modulation of enzymes and signaling kinases resulting to effects on multiple genes and cell signaling pathways. Results from clinical trials using these compounds have not shown them to be chemopreventive. This may be due to our: (1) inability to reproduce the exposure conditions, i.e., levels, complexity, other host and lifestyle factors; and (2) lack of understanding about the mechanisms of action and agent-mediated toxicity in several organs and physiological processes in the host. Current research efforts in addressing the issues of exposure conditions, bioavailability, toxicity and the mode of action of dietary phytochemicals may help address the reason for observed mismatch that may ultimately lead to identification of new chemopreventive agents for protection against broad spectrum of exposures.

Ex vivo Raman spectroscopic study of breast metastatic lesions in lungs in animal models.

The lung is one of the most common sites of metastases, with approximately 50% of patients with extrathoracic cancer exhibiting pulmonary metastases. Correct identification of the metastatic status of a lung lesion is vital to therapeutic planning and better prognosis. However, currently available diagnostic techniques, such as conventional radiography and low dose computed tomography (LDCT), may fail to identify metastatic lesions. Alternative techniques such as Raman spectroscopy (RS) are hence being extensively explored for correct diagnosis of metastasis. The current ex vivo study aims to evaluate the ability of a fiber optic-based Raman system to distinguish breast cancer metastasis in lung from primary breast and lung tumor in animal models. In this study, spectra were acquired from normal breast, primary breast tumor, normal lung, primary lung tumor, and breast cancer metastasis in lung tissues and analyzed using principal component analysis and principal component-linear discriminant analysis. Breast cancer metastasis in lung could be classified with 71% classification efficiency. Approximately 6% breast metastasis spectra were misclassified with breast tumor, probably due to the presence of breast cancer cells in metastasized lungs. Test prediction results show 64% correct prediction of breast metastasis, while 13% breast metastasis spectra were wrongly predicted as breast tumor, suggesting the possible influence of breast cancer cells. Thus, findings of this study, the first of such explorations, demonstrate the potential of RS in classifying breast metastasis in lungs from primary lung and primary breast tumor. Prospective evaluation on a larger cohort with better multivariate analysis, combined with LDCT and recently developed real-time in vivo probes, RS can play a significant role in nonsurgical screening of lesions, which can lead to individualized therapeutic regimes and improved prognoses.