The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway is frequently mutated in human cancer. This pathway consists of a small GTP protein of the RAS family that is activated in response to extracellular signaling to recruit a member of the RAF kinase family to the cell membrane. Active RAF signals through MAP/ERK kinase to activate ERK and its downstream effectors to regulate a wide range of biological activities including cell differentiation, proliferation, senescence, and survival. Mutations in the v-raf murine sarcoma viral oncogenes homolog B1 (BRAF) isoform of the RAF kinase or KRAS isoform of the RAS protein are found as activating mutations in approximately 30% of all human cancers. The BRAF pathway has become a target of interest for molecular therapy, with promising results emerging from clinical trials. Here, the role of the most common BRAF mutation BRAF^V600E in human carcinogenesis is investigated through a review of the literature, with specific focus on its role in melanoma, colorectal, and thyroid cancers and its potential as a therapeutic target. Mol Cancer Ther; 10(3); 385–94. ©2011 AACR.

Background: Archival formalin-fixed paraffin-embedded (FFPE) tissues have limited utility in applications involving analysis of gene expression due to mRNA degradation and modification during fixation and processing. This study analyzed 160 miRNAs in paired snap frozen and FFPE cells to investigate if miRNAs may be successfully detected in archival specimens. Results: Our results show that miRNA extracted from FFPE blocks was successfully amplified using Q-RT-PCR. The levels of expression of miRNA detected in total RNA extracted from FFPE were higher than that extracted from snap frozen cells when the quantity of total RNA was identical. This phenomenon is most likely explained by the fact that larger numbers of FFPE cells were required to generate equivalent quantities of total RNA than their snap frozen counterparts. Conclusion: We hypothesise that methylol cross-links between RNA and protein which occur during tissue processing inhibit the yield of total RNA. However, small RNA molecules appear to be less affected by this process and are recovered more easily in the extraction process. In general miRNAs demonstrated reliable expression levels in FFPE compared with snap frozen paired samples, suggesting these molecules might prove to be robust targets amenable to detection in archival material in the molecular pathology setting.

Aim: To analyse and compare expression patterns of three potential biomarkers—p16 ^INK4A , CDC6, and MCM5—and evaluate their use as predictive biomarkers in squamous and glandular cervical preinvasive neoplasia. Methods: Immunocytochemical analysis of p16 ^INK4A , MCM5, and CDC6 expression was performed on 20 normal, 38 cervical intraepithelial neoplasia 1 (CIN1), 33 CIN2, 46 CIN3, 10 squamous cell carcinoma, 19 cervical glandular intraepithelial neoplasia (cGIN), and 10 adenocarcinoma samples. Staining intensity was assessed using a 0–3 scoring system. p16 ^INK4A , MCM5, and CDC6 expression was also examined in ThinPrep slides exhibiting mild, moderate, and severe dyskaryosis. Human papillomavirus (HPV) was detected using a modified SYBR green assay. Fluorogenic polymerase chain reaction (PCR) and solution phase PCR were used for specific HPV typing. Results: All three markers showed a linear correlation between expression and grade of dysplasia. p16 ^INK4A and MCM5 protein expression was upregulated in all grades of squamous and glandular dysplasia. CDC6 protein was preferentially expressed in high grade lesions and in invasive squamous cell carcinoma. Conclusion: p16 ^INK4A expression was closely associated with high risk HPV infection—all grades of squamous and glandular cervical lesions were immunohistochemically positive. MCM5 staining intensity was independent of high risk HPV infection, highlighting its potential as a biomarker in both HPV dependent and independent cervical dysplasia. CDC6 may be a biomarker of high grade and invasive lesions of the cervix, with limited use in low grade dysplasia. p16 ^INK4A was the most reliable marker of cervical dysplasia. Combinations of dysplastic biomarkers may be useful in difficult diagnostic cases.

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.

Aim: To examine the potential of p16^INK4A as a biomarker for dysplastic squamous and glandular cells of the cervix in tissue sections and ThinPrep™ smears. Methods: Immunocytochemical analysis of p16^INK4A expression was performed on 22 normal cervical tissue samples, five cervical glandular intraepithelial neoplasia (cGIN), 38 cervical intraepithelial neoplasia 1 (CIN1), 33 CIN2, 46 CIN3, and 10 invasive cancer cases (eight squamous and two adenocarcinomas). All samples were formalin fixed and paraffin wax embedded, and immunohistochemical analysis was carried out using a mouse monoclonal anti-p16^INK4A antibody after antigen unmasking. The staining intensity was assessed using a 0 to 3 scoring system. In addition, the expression status of p16^INK4A was examined in 12 normal ThinPrep smears, one smear exhibiting cGIN, and a total of 20 smears exhibiting mild, moderate, and severe dyskaryosis. Human papillomavirus (HPV) detection was carried out using a modified SYBR green assay system. Fluorogenic polymerase chain reaction (PCR) and solution phase PCR were used for specific HPV typing. Results: p16^INK4A immunoreactivity was absent in all normal cervical tissues examined. Dysplastic squamous and glandular cells were positive for p16^INK4A expression in all cases included in this study, except for one CIN3 case. p16^INK4A expression was mainly nuclear in CIN1 cases, and both nuclear and cytoplasmic in CIN2, CIN3, cGIN, and invasive cases. All cases positive for HPV expressed the p16^INK4A protein, although not all cases found positive for p16^INK4A were HPV positive. In general, the p16^INK4A staining intensity was lower in cases negative for HPV or those containing a low risk HPV type. Conclusion: This pattern of overexpression demonstrates the potential use of p16^INK4A as a diagnostic marker for cervical squamous and also glandular neoplastic lesions. In addition, the technique can be used to identify individual dyskaryotic cells in ThinPrep smears. Thus, p16^INK4A is a useful marker of cervical dyskaryosis.