Background: A recently developed probe-based technology, the NanoString nCounter™ gene expression system, has been shown to allow accurate mRNA transcript quantification using low amounts of total RNA. We assessed the ability of this technology for mRNA expression quantification in archived formalin-fixed, paraffin-embedded (FFPE) oral carcinoma samples. Results: We measured the mRNA transcript abundance of 20 genes (COL3A1, COL4A1, COL5A1, COL5A2, CTHRC1, CXCL1, CXCL13, MMP1, P4HA2, PDPN, PLOD2, POSTN, SDHA, SERPINE1, SERPINE2, SERPINH1, THBS2, TNC, GAPDH, RPS18) in 38 samples (19 paired fresh-frozen and FFPE oral carcinoma tissues, archived from 1997-2008) by both NanoString and SYBR Green I fluorescent dye-based quantitative real-time PCR (RQ-PCR). We compared gene expression data obtained by NanoString vs. RQ-PCR in both fresh-frozen and FFPE samples. Fresh-frozen samples showed a good overall Pearson correlation of 0.78, and FFPE samples showed a lower overall correlation coefficient of 0.59, which is likely due to sample quality. We found a higher correlation coefficient between fresh-frozen and FFPE samples analyzed by NanoString (r = 0.90) compared to fresh-frozen and FFPE samples analyzed by RQ-PCR (r = 0.50). In addition, NanoString data showed a higher mean correlation (r = 0.94) between individual fresh-frozen and FFPE sample pairs compared to RQ-PCR (r = 0.53). Conclusions: Based on our results, we conclude that both technologies are useful for gene expression quantification in fresh-frozen or FFPE tissues; however, the probe-based NanoString method achieved superior gene expression quantification results when compared to RQ-PCR in archived FFPE samples. We believe that this newly developed technique is optimal for large-scale validation studies using total RNA isolated from archived, FFPE samples.

MicroRNAs (miRs) are non-coding RNA molecules involved in cancer initiation and progression. Deregulated miR expression has been implicated in cancer; however, there are no studies implicating an miR signature associated with progression in oral squamous cell carcinoma (OSCC). Although OSCC may develop from oral leukoplakia, clinical and histological assessments have limited prognostic value in predicting which leukoplakic lesions will progress. Our aim was to quantify miR expression changes in leukoplakia and same-site OSCC and to identify an miR signature associated with progression. We examined miR expression changes in 43 sequential progressive samples from 12 patients and four non-progressive leukoplakias from four different patients, using TaqMan Low Density Arrays. The findings were validated using quantitative RT-PCR in an independent cohort of 52 progressive dysplasias and OSCCs, and five non-progressive dysplasias. Global miR expression profiles distinguished progressive leukoplakia/OSCC from non-progressive leukoplakias/normal tissues. One hundred and nine miRs were highly expressed exclusively in progressive leukoplakia and invasive OSCC. miR-21, miR-181b and miR-345 expressions were consistently increased and associated with increases in lesion severity during progression. Over-expression of miR-21, miR-181b and miR-345 may play an important role in malignant transformation. Our study provides the first evidence of an miR signature potentially useful for identifying leukoplakias at risk of malignant transformation.

Background: Oral Squamous Cell Carcinoma (OSCC) is a major cause of cancer death worldwide, which is mainly due to recurrence leading to treatment failure and patient death. Histological status of surgical margins is a currently available assessment for recurrence risk in OSCC; however histological status does not predict recurrence, even in patients with histologically negative margins. Therefore, molecular analysis of histologically normal resection margins and the corresponding OSCC may aid in identifying a gene signature predictive of recurrence.Methods: We used a meta-analysis of 199 samples (OSCCs and normal oral tissues) from five public microarray datasets, in addition to our microarray analysis of 96 OSCCs and histologically normal margins from 24 patients, to train a gene signature for recurrence. Validation was performed by quantitative real-time PCR using 136 samples from an independent cohort of 30 patients.Results: We identified 138 significantly over-expressed genes (> 2-fold, false discovery rate of 0.01) in OSCC. By penalized likelihood Cox regression, we identified a 4-gene signature with prognostic value for recurrence in our training set. This signature comprised the invasion-related genesMMP1,COL4A1,P4HA2, andTHBS2. Over-expression of this 4-gene signature in histologically normal margins was associated with recurrence in our training cohort (p = 0.0003, logrank test) and in our independent validation cohort (p = 0.04, HR = 6.8, logrank test).Conclusion: Gene expression alterations occur in histologically normal margins in OSCC. Over-expression of the 4-gene signature in histologically normal surgical margins was validated and highly predictive of recurrence in an independent patient cohort. Our findings may be applied to develop a molecular test, which would be clinically useful to help predict which patients are at a higher risk of local recurrence.

Lung cancer and chronic obstructive pulmonary disease (COPD) often co-occur, and individuals with COPD are at a higher risk of developing lung cancer. While the underlying mechanism for this risk is not well understood, its major contributing factors have been proposed to include genomic, immune, and microenvironment dysregulation. Here, we review the evidence and significant studies that explore the mechanisms underlying the heightened lung cancer risk in people with COPD. Genetic and epigenetic changes, as well as the aberrant expression of non-coding RNAs, predispose the lung epithelium to carcinogenesis by altering the expression of cancer- and immune-related genes. Oxidative stress generated by tobacco smoking plays a role in reducing genomic integrity, promoting epithelial-mesenchymal-transition, and generating a chronic inflammatory environment. This leads to abnormal immune responses that promote cancer development, though not all smokers develop lung cancer. Sex differences in the metabolism of tobacco smoke predispose females to developing COPD and accumulating damage from oxidative stress that poses a risk for the development of lung cancer. Dysregulation of the lung microenvironment and microbiome contributes to chronic inflammation, which is observed in COPD and known to facilitate cancer initiation in various tumor types. Further, there is a need to better characterize and identify the proportion of individuals with COPD who are at a high risk for developing lung cancer. We evaluate possible novel and individualized screening strategies, including biomarkers identified in genetic studies and exhaled breath condensate analysis. We also discuss the use of corticosteroids and statins as chemopreventive agents to prevent lung cancer. It is crucial that we optimize the current methods for the early detection and management of lung cancer and COPD in order to improve the health outcomes for a large affected population.

Background: The tumor suppressor Programmed Cell Death 4 (PDCD4) has been found to be under-expressed in several cancers and associated with disease progression and metastasis. There are no current studies characterizing PDCD4 expression and its clinical relevance in Oral Squamous Cell Carcinoma (OSCC). Since nodal metastasis is a major prognostic factor in OSCC, we focused on determining whether PDCD4 under-expression was associated with patient nodal status and had functional relevance in OSCC invasion. We also examinedPDCD4regulation by microRNA 21 (miR-21) in OSCC.Results: PDCD4mRNA expression levels were assessed in 50 OSCCs and 25 normal oral tissues.PDCD4was under-expressed in 43/50 (86%) OSCCs, with significantly reduced mRNA levels in patients with nodal metastasis (p = 0.0027), and marginally associated with T3-T4 tumor stage (p = 0.054). PDCD4 protein expression was assessed, by immunohistochemistry (IHC), in 28/50 OSCCs and adjacent normal tissues; PDCD4 protein was absent/under-expressed in 25/28 (89%) OSCCs, and marginally associated with nodal metastasis (p = 0.059). A matrigel invasion assay showed that PDCD4 expression suppressed invasion, and siRNA-mediated PDCD4 loss was associated with increased invasive potential of oral carcinoma cells. Furthermore, we showed that miR-21 levels were increased in PDCD4-negative tumors, and thatPDCD4expression may be down-regulated in OSCC by direct binding of miR-21 to the 3'UTRPDCD4mRNA.Conclusions: Our data show an association between the loss of PDCD4 expression, tumorigenesis and invasion in OSCC, and also identify a mechanism of PDCD4 down-regulation by microRNA-21 in oral carcinoma. PDCD4 association with nodal metastasis and invasion suggests that PDCD4 may be a clinically relevant biomarker with prognostic value in OSCC.

BACKGROUND.: The authors have previously shown that overexpression of claudin 1 (CLDN1) is associated with advanced disease stage in oral squamous cell carcinomas (OSCCs). Their goal was to examine CLDN1 expression in a large series of primary OSCCs and to further investigate whether CLDN1 overexpression plays a role in invasion in OSCC.METHODS.: CLDN1 gene expression levels were determined by quantitative real‐time reverse transcription polymerase chain reaction (QRT‐PCR) in 100 primary OSCCs. CLDN1 protein expression was examined by immunohistochemistry in 70 of 100 OSCCs. E‐Cadherin protein levels were also assessed in 58 OSCCs. The authors performed a transwell Matrigel invasion assay for assessment of the invasive potential of CLDN1 overexpressing oral carcinoma cells. Western blotting and QRT‐PCR were used to assess CLDN1 expression in transfected cells and controls.RESULTS.: CLDN1 mRNA was increased (median = 18.5) in 79 of 100 OSCCs, compared with normal oral mucosa (expression = 1.0). CLDN1 overexpression was associated with angiolymphatic (P = .037) and perineural invasion (P = .051). CLDN1 was highly expressed in 48 of 70 (68%) OSCCs. E‐Cadherin was lost or underexpressed in 49 of 58 (84%) OSCCs. The invasion assay showed that cells overexpressing CLDN1 have increased invasive potential, whereas small interfering RNA–mediated depletion of CLDN1 decreased the invasive potential of cells.CONCLUSIONS.: CLDN1 overexpression is associated with angiolymphatic and perineural invasion, consistent with aggressive tumor behavior. Overexpression of CLDN1 protein is associated with increased invasiveness of oral carcinoma cells. Cancer 2008. © 2008 American Cancer Society.