Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring.

Personalized medicine in oncology needs immunological test standards. Flow cytometry (FCM) method is an important tool for immunomonitoring, and their harmonization is essential to obtain comparable data in multicenter clinical trials. The purpose of this study was to design a workflow harmonization can address the problems most effectively contribute to intra- and inter-operator variability in project.

The multicenter Italian National Institute of Health (Istituto Superiore di Sanità, ISS) succeeded in multiparametric flow cytometric panel harmonization between thirteen owned operator of five clinical centers and research areas Lazio (Italy). panel based on a mixture backbone of antibodies dried (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA and anti-CCR7) for detecting cell / memory T naive, is recognized as a potential prognostic / predictive immunological biomarkers in immunotherapies of cancer.

The coordination center congealed distributed peripheral blood mononuclear cells (PBMC) and a fresh sample of whole blood (WB) from a healthy donor, reagents and standard operating procedures (SOP) for participants to experiment with their own equipment, to simulate a real-life scenario. Operators and local back raw data were analyzed for the ISS for central analysis and statistics elaboration.Harmonised and reproducible results are obtained by sharing the experimental set-up and procedures along with the centralization of data analysis, which leads to a reduction in cross-center variability naive / memory section in particular frequency whole blood setting.

Our experimental and analytical work processes proved to be suitable for harmonization of FCM test in a multicenter setting, where high quality data needed to evaluate the potential for immunological markers, which can contribute to choose a better therapeutic selection.

 Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring.
Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring.

RNAseq in comparison to Immunohistochemistry to Measure Cancer Biomarkers in Breast Cancer and Lung Cancer Specimens.

RNA sequencing is considered the gold standard for high-throughput gene expression profile at the level of transcription. increasingly important in cancer research and molecular diagnostics is reflected in the growing number of mentions in the scientific literature and clinical trials reports. However, the use of different reagents and protocols for RNA sequencing often produce results that are not compatible.

More recently, we published a profile of Atlas Oncobox RNA sequencing to normal human tissues were obtained from a healthy donor were killed in road accidents. This is a database of molecular profiles obtained using the setting uniform protocol and reagents that can be widely used in biomedicine for the normalization of data in pathology, including cancer.

Here, we publish 39 original new breast cancer (BC) and the profile 19 of lung cancer (LC) RNA sequencing was obtained for paraffin-embedded samples (FFPE) tissue formalin-fixed, fully compatible with Oncobox Atlas. We did the first correlation study RNA sequencing and immunohistochemical expression profile-measured for actionable clinical biomarker genes in the cancer FFPE tissue samples. We showed a high (Spearman’s rho 0.65 to 0.798) and statistically significant (p <0.00004) correlation between RNA sequencing (Oncobox protocol) and immunohistochemical measurement of HER2 / ERBB2, ER / ESR1 and PGR gene in BC, and for gene PDL1 The LC; AUC: 0.963 to HER2, 0.921 for ESR1, 0.912 to PGR, and 0.922 for PDL1.

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To our knowledge, this is the first validation total RNA sequencing archived FFPE material provide a reliable estimate of the level of the marker protein. These results suggest that in the future, RNA sequencing can equip immunohistochemistry for the reliable measurement of biomarker expression in FFPE cancer samples.