Tumor histoculture captures the dynamic interactions between tumor and immune components in response to anti-PD1 in head and neck cancer.

Dynamic interactions within the tumor micro-environment drive patient response to immune checkpoint inhibitors. Existing preclinical models lack true representation of this complexity. Using a Head and Neck cancer patient derived TruTumor histoculture platform, the response spectrum of 70 patients to anti-PD1 treatment is investigated in this study. With a subset of 55 patient samples, multiple assays to characterize T-cell reinvigoration and tumor cytotoxicity are performed. Based on levels of these two response parameters, patients are stratified into five sub-cohorts, with the best responder and non-responder sub-cohorts falling at extreme ends of the spectrum. The responder sub-cohort exhibits high T-cell reinvigoration, high tumor cytotoxicity with T-cells homing into the tumor upon treatment whereas immune suppression and tumor progression pathways are pre-dominant in the non-responders. Some moderate responders benefit from combination of anti-CTLA4 with anti-PD1, which is evident from better cytotoxic T-cell: T-regulatory cell ratio and enhancement of tumor cytotoxicity. Baseline and on-treatment gene expression signatures from this study stratify responders and non-responders in unrelated clinical datasets.

Androgen Receptor mRNA levels determine the prognosis in triple-negative breast cancer patients.

BACKGROUND: Anti-Androgen Receptor (AR) therapy holds promise for a subset of AR expressing triple-negative breast cancer (TNBC) patients. However, current AR assays are suboptimal in detecting the dynamic range of AR expression, contributing to its controversial role in TNBC disease prognosis. This study is aimed at evaluating the feasibility of qRT-PCR to sensitively and robustly detect AR mRNA levels for prognostication. METHODS: mRNA expression profiling was performed on FFPE blocks from a retrospective cohort of 101 TNBC patients using qRT-PCR and compared with AR protein expression by immunohistochemistry . Statistical analyses included Spearman's rank correlation, Chi-square and Kaplan-Meier analyses. Distant Metastasis Free Survival was used as the end point in survival analysis. RESULTS: AR mRNA expression was observed in 34/101 patients (34%) whereas 12/80 cases (15%) were positive by IHC. qRT-PCR could thus detect more AR positive patients as compared to IHC, with 75% (9/12) concordance between the two methods. Co-expression of GATA3 and FOXA1 mRNA was observed in 85 and 88% of AR mRNA positive tumors, respectively. AR mRNA positivity was significantly correlated with age at disease onset (p = 0.02), high FOXA1/GATA3 (p < 0.05) and distant recurrence. AR mRNA positive patients had poorer DMFS (43%; p = 0.002). DMFS dropped further to 26% (p = 0.006) in AR (+)/high FOXA1/GATA3 patients. AR mRNA expression together with node positivity had the worst DMFS (23%; p < 0.0001) compared to patients who were either positive for any one of these, or negative for both AR and node status. Low Ki67 mRNA with AR mRNA positivity also had poorer DMFS (39%; p = 0.001) compared to patients expressing low Ki67 with no AR mRNA expression. CONCLUSION: qRT-PCR was more sensitive and reliable in detecting the dynamic expression levels of AR compared to IHC and this variation could be explained by the higher sensitivity of the former method. High AR mRNA expression was strongly associated with expression of AR protein, high FOXA1/GATA3 mRNA, and with poor prognosis. qRT-PCR was more efficient in detecting the AR positive cases compared to IHC. A distinct signature involving high GATA3/FOXA1, low Ki67, and node positivity in AR mRNA positive tumors correlated with poor prognosis. Thus, AR mRNA screening can serve as an effective prognostic marker along with offering potential targeted therapy options for TNBC.

Cancer stem cell and its niche in malignant progression of oral potentially malignant disorders.

OBJECTIVE: The purpose of this study was to determine association between cancer stem cells (CSCs) and their niche with progression of oral potentially malignant disorders. MATERIALS AND METHODS: Patients with histologically confirmed oral potentially malignant disorders, stratified into high/low risk lesions based on the degree of dysplasia and oral cancer were included in this study. Immunohistochemical profiling of markers of CSCs (CD44), endothelial cells (CD31) and CSC-vascular niche cross-talk (CXCR4 and SDF1) were carried out. Statistical analysis was performed to correlate the relationship of markers with histopathology grade (ANOVA, and χ2 test, unpaired t test) using GraphPad InStat v3.06. RESULTS: The study included 550 samples (349 patients) and analysis showed progressive increase in expression levels of CSC and its niche markers with increase in grade of dysplasia as compared to the normal cohort (p < 0.05). Co-expression analysis revealed that, in comparison to the normal cohort, a larger percentage of patients showed increased expression of CD31 and CD44 (CD31high/CD44high; p < 0.05) and of CXCR4 and SDF1 (CXCR4high/SDF1high; p = 0.04), suggesting an association of the CSCs and the vascular niche. Further, distribution of patients with CD44high/CXCR4high (p < 0.05) and CD31high/SDF1high (p = 0.01) was significantly increased in the high-risk group (18%), suggesting a correlation between CD44+/CXCR4+ cells, the vascular niche and progression of oral dysplastic lesions. CONCLUSION: The increased expression of CSCs, the vascular niche and their cross talk markers are associated with increase in severity of dysplasia suggesting their role in the progression of oral potentially malignant disorders and may hence be used in identifying high-risk OPMD.

Analysis of the invasive edge in primary and secondary oral squamous cell carcinoma: An independent prognostic marker: A retrospective study.

BACKGROUND AND OBJECTIVES: Oral squamous cell carcinoma (OSCC) is one of the most common head and neck carcinomas and corresponds to 95% of all oral cancers with an increasing morbidity and mortality. Its prognosis is affected by several clinicopathologic factors, one of which is pattern of invasion (POI). The histological features of OSCC may differ widely, but there is general agreement that the most useful prognostic information can be deduced from the invasive front of the tumor. In this retrospective study, our aim was to compare the POI, the status of connective tissue and the status of inflammation at the tumor-host interface in primary and recurrent (secondary) OSCC and test the validity of POI, to serve as a potential marker to assess the prognosis of the patient. MATERIALS AND METHODS: Differentiation of tumors, POI, status of connective tissue and inflammation was assessed in 168 cases of primary and recurrent cases of OSCC. STATISTICAL ANALYSIS: Fisher's exact test was used to determine the statistical significance and P < 0.05 was considered to be statistically significant. RESULTS: Our study showed that majority of the primary and secondary tumors were well differentiated, 117 [95.9%] and 34 [73.9%], respectively. Predominant POI in the primary and secondary tumor group was Pattern II and least was Pattern V. Worst pattern in primary tumor and highest distribution was seen for Pattern III (53.3%), and least for Pattern V (0.00%). In secondary tumors, the predominant worst pattern was Pattern IV (50.0%) and least distribution was seen for Pattern I (0.00%). Connective tissue status for both primary and secondary tumors showed the predominance of loose type (85.2% and 79.2%) and least was variable type (0.8% and 0.6%), respectively. Status of inflammation in the primary tumor group showed a predominance of moderate grade of inflammation (50.0%) and very mild grade of inflammation (6.6%) was the least type. In the secondary tumor group, moderate grade (43.5%) of inflammation was predominant and very mild grade (5.4%) was the least. All the parameters showed a statistically significant difference on the application of Fisher's exact test between the two groups. CONCLUSION: Our study showed that POI could serve as an individual prognostic marker irrespective of the histologic differentiation of tumor. Tumor desmoplasia could be considered as an important reflection of the tumor-host interaction, especially in aggressive cancers. Host immune defense, especially tumor infiltrating lymphocytes must be noted as critical factors related to survival rate in OSCC patients. Assessment of mentioned parameters may lead to sound prognostic assessment and appropriate treatment planning thus reducing the possibility of recurrence or relapse. Hence, the parameters evaluated in our study could serve as independent or interdependent prognostic markers.

Meta-Analyses of Microarray Datasets Identifies ANO1 and FADD as Prognostic Markers of Head and Neck Cancer.

The head and neck squamous cell carcinoma (HNSCC) transcriptome has been profiled extensively, nevertheless, identifying biomarkers that are clinically relevant and thereby with translational benefit, has been a major challenge. The objective of this study was to use a meta-analysis based approach to catalog candidate biomarkers with high potential for clinical application in HNSCC. Data from publically available microarray series (N = 20) profiled using Agilent (4X44K G4112F) and Affymetrix (HGU133A, U133A_2, U133Plus 2) platforms was downloaded and analyzed in a platform/chip-specific manner (GeneSpring software v12.5, Agilent, USA). Principal Component Analysis (PCA) and clustering analysis was carried out iteratively for segregating outliers; 140 normal and 277 tumor samples from 15 series were included in the final analysis. The analyses identified 181 differentially expressed, concordant and statistically significant genes; STRING analysis revealed interactions between 122 of them, with two major gene clusters connected by multiple nodes (MYC, FOS and HSPA4). Validation in the HNSCC-specific database (N = 528) in The Cancer Genome Atlas (TCGA) identified a panel (ECT2, ANO1, TP63, FADD, EXT1, NCBP2) that was altered in 30% of the samples. Validation in treatment naïve (Group I; N = 12) and post treatment (Group II; N = 12) patients identified 8 genes significantly associated with the disease (Area under curve>0.6). Correlation with recurrence/re-recurrence showed ANO1 had highest efficacy (sensitivity: 0.8, specificity: 0.6) to predict failure in Group I. UBE2V2, PLAC8, FADD and TTK showed high sensitivity (1.00) in Group I while UBE2V2 and CRYM were highly sensitive (>0.8) in predicting re-recurrence in Group II. Further, TCGA analysis showed that ANO1 and FADD, located at 11q13, were co-expressed at transcript level and significantly associated with overall and disease-free survival (p<0.05). The meta-analysis approach adopted in this study has identified candidate markers correlated with disease outcome in HNSCC; further validation in a larger cohort of patients will establish their clinical relevance.

Establishment and characterization of triple drug resistant head and neck squamous cell carcinoma cell lines.

Resistance to chemotherapy leading to poor outcome and survival remains a challenge for developing strategies for therapeutic interventions in all types of cancer, including head and neck cancer. In vitro chemoresistant cell line models are an indispensable resource towards delineating the mechanisms involved in drug resistance/response and for the development of novel drugs. Current treatment for head and neck cancer includes chemotherapy with cisplatin, docetaxel and 5-fluorouracil (5-FU) and the response rates to these drugs in patients is 60-80%. The present study aimed to generate head and neck cancer triple drug-resistant cell lines in an effort towards elucidating the mechanisms underlying chemoresistance and providing a resourceful tool for drug design. Using two head and neck squamous cell carcinoma cell lines, Hep-2 (larynx) and CAL-27 (oral cavity), the present study sequentially exposed these cells to increasing concentrations of the combination of docetaxel, cisplatin and 5-FU (TPF) to generate triple drug-resistant cells, termed Hep-2 TPF resistant (TPFR) and CAL-27 TPFR. The effect of the drug treatments on the cell viability, apoptosis, cell cycle and the expression of genes associated with multidrug resistance were analyzed in the parental cells and drug-resistant counterparts.

Factors predicting outcome after salvage treatment for stage IV oral squamous cell carcinoma: Evidence of the potential importance of the cyclooxygenase-2-prostaglandin E2 pathway.

BACKGROUND: We determined the clinicopathological factors that predicted outcome after salvage treatment for stage IV oral squamous cell carcinoma (OSCC). Additionally, the prognostic significance of the cyclooxygenase-2 (COX-2)/microsomal prostaglandin-E synthase-1 (mPGES-1) pathway was evaluated. METHODS: Thirty-one patients who underwent salvage surgery were included. COX-2 and mPGES-1 levels were quantified by real time polymerase chain reaction (PCR). RESULTS: The 2-year disease-free and overall survival rates were 46% and 53%, respectively. Adequacy of initial treatment, tobacco smoking, and the presence of pathological risk factors were predictive of mortality. In patients who had not received chemotherapy before salvage surgery, high levels of intratumoral COX-2 and mPGES-1 were associated with poor prognosis. By contrast, high intratumoral COX-2 and mPGES-1 after chemotherapy were associated with improved outcomes. CONCLUSION: Clinicopathological factors may inform treatment decisions in patients with stage IV OSCC. Expression patterns of COX-2 and mPGES-1 correlated with outcome and warrant further investigation. © 2014 Wiley Periodicals, Inc. Head Neck 37: 1142-1149, 2015.