OBJECTIVE.—: To validate Anyplex II HPV HR Detection (Anyplex II, Seegene) using FFPE samples.
DESIGN.—: We used 248 stored DNA extracts from cervical cancer FFPE samples collected during 2005-2015 that tested HPV positive using the RHA kit HPV SPF10-LiPA25, v1 (SPF10, Labo Biomedical Products) HPV genotyping assay, manufacturer-validated for FFPE samples.
RESULTS.—: Of the selected 248 samples, 243 were used in our analysis. Consistent with SPF10 genotyping results, Anyplex II detected all 12 oncogenic types and had an overall HPV detection rate of 86.4% (210 of 243 samples). Anyplex II and SPF10 showed very high agreement for the detection of the 2 most important oncogenic genotypes: HPV 16 (219 of 226; 96.9%; 95% CI, 93.7-98.75) and HPV 18 (221 of 226; 97.8%; 95% CI, 94.9-99.3).
CONCLUSIONS.—: Overall results showed that both platforms produced comparable HPV genotyping results, indicating the suitability of Anyplex II for FFPE samples. The Anyplex II assay has the added convenience of being an efficient, single-well semiquantitative polymerase chain reaction assay. Further optimization of Anyplex II may enhance its performance using FFPE samples by improving the detection limit.
Materials and Methods: Biodegradable polymeric microneedle arrays were fabricated out of poly lactic-co-glycolic acid (PLGA) using the micromolding technique under aseptic conditions, and the morphology of the microneedles was characterized using light microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to rule out drug-polymer interactions. Standard procedures were used to analyze the prepared microneedle arrays for in vitro drug release and to perform a microneedle insertion test. Enzyme-linked immunosorbent assay was used to quantify rHuKGF.
Results: The PLGA polymer was safe for use in the fabrication of rHuKGF microneedles as there was no interaction between the drug and the polymer. The fabricated rHuKGF microneedle arrays had fully formed microneedles with a height of 600 µm and a base of 300 µm. The drug from the microneedle patch was released in vitro within 30 minutes. The strength of the microneedles in the patch was good, as they were able to reach a depth of 381±3.56 µm into parafilm without any structural change or fracture.
Conclusion: Microneedle transdermal patches were successfully prepared for rHuKGF, and their evaluation suggested excellent quality and uniformity of patch characteristics. This can have potential applications in the therapeutic arena, offering advantages in terms of reduced dosing frequency, improved patient compliance, and bioavailability.
METHODS: ALK gene rearrangement was detected by immunostaining of ALK protein and fluorescence in situ hybridisation (FISH) targeting at the 2p23 region.
RESULTS: The expression of ALK protein was detected in 24/34 (71%) of the cases, and it was significantly higher in childhood cases (100%) when compared to adult cases (47%). The analyses by FISH were consistent with the results from immunostaining of ALK protein, but the analyses were only successful in 15/34 (44%) cases. FISH analyses detected extra copies of ALK gene in three cases, including one case that expressed ALK protein and showed 2p23 rearrangement.
CONCLUSIONS: The current series revealed a high frequency of ALK gene rearrangement, especially in the children. Immunostaining of ALK protein is a reliable indication of ALK gene rearrangement, and is superior to FISH. However, FISH analysis is useful in detecting other genetic aberrations that are not related to ALK gene rearrangement.
METHODS: Sprague Dawley rats (n = 7, body weight = 300 g ± 50 g) were grouped randomly into two groups-control (n = 3) and expanded (n = 4). Anisotropic hydrogel tissue expanders were inserted into the frontal maxillofacial region of the rats in the expanded group. The rats were sacrificed, and skin samples were harvested, fixed in formalin, and embedded in paraffin wax for histological investigation. Hematoxylin and eosin staining was performed to detect histological changes between the two groups and to investigate the inflammatory response in the expanded samples. Three inflammatory markers, namely interleukin (IL)-1α, IL-6, and tumor necrosis factor-α (TNF-α), were analyzed by immunohistochemistry.
RESULT: IL-1-α expression was only observed in the expanded tissue samples compared to the controls. In contrast, there was no significant difference in IL-6, and TNF-α production. Histological analysis showed the absence of inflammatory response in expanded tissues, and a negative non-significant correlation (Spearman's correlation coefficient) between IL-1-α immune-positive cells and the inflammatory cells (r = -0.500). In conclusion, tissues that are expanded and stabilized using an anisotropic self-inflating hydrogel tissue expander might be useful for tissue replacement and engraftment as the expanded tissue does not show any sign of inflammatory responses. Detection of IL-1-α in the expanded tissues warrants further investigation for its involvement without any visible inflammatory response.
METHODS: Patients with oral epithelial dysplasia at one hospital were selected as the 'training set' (n = 56) whilst those at another hospital were selected for the 'test set' (n = 66). RNA was extracted from formalin-fixed paraffin-embedded (FFPE) diagnostic biopsies and analysed using the NanoString nCounter platform. A targeted panel of 42 genes selected on their association with oral carcinogenesis was used to develop a prognostic gene signature. Following data normalisation, uni- and multivariable analysis, as well as prognostic modelling, were employed to develop and validate the gene signature.
RESULTS: A prognostic classifier composed of 11 genes was developed using the training set. The multivariable prognostic model was used to predict patient risk scores in the test set. The prognostic gene signature was an independent predictor of malignant transformation when assessed in the test set, with the high-risk group showing worse prognosis [Hazard ratio = 12.65, p = 0.0003].
CONCLUSIONS: This study demonstrates proof of principle that RNA extracted from FFPE diagnostic biopsies of OPMD, when analysed on the NanoString nCounter platform, can be used to generate a molecular classifier that stratifies the risk of malignant transformation with promising clinical utility.