sybr-green-i and Carcinoma--Squamous-Cell

Human kallikrein 11 gene (KLK11) encodes a secreted serine protease. In view of its diagnostic and prognostic strength in many malignancies, we investigated the mRNA expression levels of KLK11 in laryngeal tissues in order to unveil its clinical usefulness in laryngeal cancer.. KLK11 expression was quantified in 163 tissue samples from 105 laryngeal cancer patients with the development of a highly sensitive real-time PCR methodology, using SYBR Green® chemistry.. KLK11 expression in laryngeal cancer specimens of primary or recurrent nature was significantly inferior compared with their non-malignant counterparts (P<0.001 and P=0.026, respectively), a finding of immense diagnostic value as illustrated in the ROC curve analyses (P<0.001). Survival analysis showed that patients harboring KLK11-positive tumors had a significantly decreased risk of death (HR=0.26, P=0.042).. Our data recommend KLK11 mRNA expression as a novel and independent biomarker in laryngeal cancer for diagnostic and prognostic purposes.

Topics: Adult; Aged; Aged, 80 and over; Benzothiazoles; Biomarkers, Tumor; Carcinoma, Squamous Cell; Diamines; Female; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Humans; Laryngeal Neoplasms; Male; Middle Aged; Organic Chemicals; Prognosis; Quinolines; Real-Time Polymerase Chain Reaction; Retrospective Studies; RNA, Messenger; ROC Curve; Serine Endopeptidases; Survival Analysis

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.. 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).. 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.

Topics: Benzothiazoles; Carcinoma, Squamous Cell; Color; Diamines; Fluorescent Dyes; Gene Expression Profiling; Humans; Molecular Probe Techniques; Mouth Neoplasms; Nanotechnology; Nucleic Acid Probes; Organic Chemicals; Paraffin Embedding; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Human papilloma virus (HPV) load and physical status are considered useful parameters for clinical evaluation of cervical squamous cell neoplasia. However, the errors implicit in HPV gene quantification by PCR are not well documented. We have undertaken the first rigorous evaluation of the errors that can be expected when using SYBR green qPCR for quantification of HPV type 16 gene copy numbers. We assessed a modified method, in which external calibration curves were generated from a single construct containing HPV16 E2, HPV16 E6 and the host gene hydroxymethylbilane synthase in a 1:1:1 ratio.. When testing dilutions of mixed HPV/host DNA in replicate runs, we observed errors in quantifying E2 and E6 amplicons of 5-40%, with greatest error at the lowest DNA template concentration (3 ng/microl). Errors in determining viral copy numbers per diploid genome were 13-53%. Nevertheless, in cervical keratinocyte cell lines we observed reasonable agreement between viral loads determined by qPCR and Southern blotting. The mean E2/E6 ratio in episome-only cells was 1.04, but with a range of 0.76-1.32. In three integrant-only lines the mean E2/E6 ratios were 0.20, 0.72 and 2.61 (values confirmed by gene-specific Southern blotting). When E2/E6 ratios in fourteen HPV16-positive cervical carcinomas were analysed, conclusions regarding viral physical state could only be made in three cases, where the E2/E6 ratio was < or = 0.06.. Run-to-run variation in SYBR green qPCR produces unavoidable inaccuracies that should be allowed for when quantifying HPV gene copy number. While E6 copy numbers can be considered to provide a useable indication of viral loads, the E2/E6 ratio is of limited value. Previous studies may have overestimated the frequency of mixed episomal/integrant HPV infections.

Topics: Base Sequence; Benzothiazoles; Calibration; Carcinoma, Squamous Cell; Cell Line, Tumor; Diamines; DNA-Binding Proteins; DNA, Viral; Female; Gene Dosage; Human papillomavirus 16; Humans; Hydroxymethylbilane Synthase; Molecular Sequence Data; Oncogene Proteins, Viral; Organic Chemicals; Papillomavirus Infections; Polymerase Chain Reaction; Quinolines; Repressor Proteins; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms; Viral Load

Cyclooxygenase-2 (COX-2) displays anti-apoptotic functions related to angiogenesis or blocking of bcl-2 functions. COX-2 overexpression has been found in various human malignancies, including esophageal squamous cell carcinoma (ESCC). The present study examined correlations between expression of COX-2 mRNA and ESCC responses to chemoradiotherapy (CRT). Expression of COX-2 mRNA obtained from 29 biopsy specimens before CRT was quantified using reverse transcriptase-polymerase chain reaction (RT-PCR) using Sybr Green I on the Roche LightCycler system. CRT comprised 5-fluorouracil plus cisplatin and 40 Gy of radiation. Mean COX-2 mRNA score was significantly higher in tumors (1222) than in normal epithelium (50; p=0.05). Expression of COX-2 mRNA was high in 14 patients, low in 8 and absent in 7. The effective response to CRT was achieved in 18 patients. Mean COX-2 mRNA score was significantly higher in ineffective cases (2910) than in effective cases (190; p<0.05). CRT was more effective in patients with low COX-2 mRNA expression than in those with high expression. COX-2 mRNA expression in biopsy specimens was closely related to CRT effectiveness. Examination of COX-2 mRNA expression is useful for predicting the effect of CRT in patients with ESCC.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Benzothiazoles; Biopsy; Calibration; Carcinoma, Squamous Cell; Cell Line, Tumor; Cisplatin; Combined Modality Therapy; Cyclooxygenase 2; Diamines; DNA, Complementary; Esophageal Neoplasms; Female; Fluorescent Dyes; Fluorouracil; Humans; Male; Membrane Proteins; Middle Aged; Neoplasm Metastasis; Organic Chemicals; Prognosis; Prostaglandin-Endoperoxide Synthases; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Time Factors; Treatment Outcome

In this study we developed an in situ protocol for quantitative detection of high-risk human papillomavirus (HPV), based on direct in situ polymerase chain reaction (PCR) with SYBR Green I labeling and GeneAmp 5700 Sequence Detection System technology. This protocol was applied on cytological specimens of patients with cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma (SCC). We performed direct in situ quantitative PCR on cell smears, uninfected human skin fibroblasts, Hela and Caski cells. After in situ amplification, slides were counterstained with propidium iodide and analyzed under a fluorescent microscope in order to localize high-risk HPV and verify preservation of morphology. After PCR optimization, we obtained the following results. The Hela cells showed values ranging from 15 to 33 copies of high-risk HPV per cell, the Caski cell line from 220 to 300 high-risk HPV copies per cell and the cell smear (both CIN and SCC) around 20-35 copies of high-risk HPV per cell. No high-risk HPV amplification was detected in uninfected human fibroblasts, healthy controls, non-amplification control, and non-specific primer control. A positive intranuclear high-risk HPV amplification was detected in cell smears from 20 patients with CIN and 10 with SCC. In conclusion, our in situ quantitative protocol for high-risk HPV detection on cell smears combines both quantitative data and in situ localization of the target, with preservation of morphology. For this reason it could be used as a rapid screening tool when both morphological and quantitative results are requested on the same slide.

Topics: Benzothiazoles; Carcinoma, Squamous Cell; Diamines; Female; Fluorescent Dyes; HeLa Cells; Humans; In Situ Hybridization, Fluorescence; Organic Chemicals; Papillomaviridae; Polymerase Chain Reaction; Quinolines; Sensitivity and Specificity; Tumor Cells, Cultured; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms