Exact characterization the The tumor microenvironment when the tissue sample is accessible restricted Maybe key The challenge in immuno-oncology area. The scale and tumor infiltration of T.–Cellular populations are from prominent interest. In this interview, NewsMedical Talking to Sirpa search on Use and value of regulatory immunohistochemical multiplexes for analytical validation of solid tumour.
What are T cells and why are they important?
T cells are usually classified as helper (Th), cytotoxic (Tcyto), memory cells, or regulatory (Treg) cells. T cells are important immune cells, making them favored targets for immune modulation. Tcyto cells provide optimal immune responses and protect against microbial invasion and tumors antigens. Under homeostatic conditions, Tregs promote peripheral tolerance. However, when it comes to tumors, Tregs may suppress Tcyto-cell functions.
What is the multiplexing protocol and how was it developed?
Cerba Research developed the multiplexing protocol, histoprofile®– T-reg light panel, using the Discovery ULTRA platform (Ventana), which was designed to stain specific T-cell subsets on a single slide. This precluded the need for serial sections of precious formalin-fixed, formalin-fixed (FFPE) patient samples in clinical trials while still providing a comprehensive analysis of the tumor microenvironment.
The multiplex protocol has been optimized and previously validated on a non-small cell lung cancer FFPE sample. Analytical validation included tests of specificity, sensitivity, accuracy, and antigen stability on healthy tonsils (control) and pathological FFPE samples of breast, lung, head and neck, and colon obtained from the Cerba Research Montpellier Biobank.
Can you describe a specific application that includes Histoprofile®T-reg light board multiplexing protocol?
Histoprofile evaluation®Specificity of the T-reg light protocol, a multiplex protocol was used to investigate healthy human amygdala samples (negative/positive controls). The FDA-approved Healthy Multi-Tissue TMA is made from 33 different tissues from three donors. In this application, the pathologist verified the specificity of the three biomarkers on all tissues tested.
histoprofile®The Tregs Mild protocol has been tested on a range of FFPE samples for solid tumors, including non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), head and neck squamous cell carcinoma (HNSCC), and colorectal cancer (CRC). ) ).
Complement TMA multi-organ tumor tissue blocks to evaluate a sufficient number of samples for each indication. The pathologist then recorded slides to give a semi-quantitative analysis of targets and confirm identification.
How is the analytical accuracy of Histoprofile®– Light T-reg plate rating?
To assess the analytical accuracy of Histoprofile®-T-reg light protocol, we performed intraoperative reproducibility and interoperability on two breast, lung, colorectal, and head and neck cancer samples. In this assessment, slides were analyzed using Halo for cell density. The CVs of all samples were then averaged for each solid tumor and tissue type.
When the densities of cells positive for CD3, CD8, and FoxP3 are used as a readout of the protocol, it is possible to meet the acceptance criteria (CV<20%) for solid tumors. For CD3 the CV is 21%, but at Cerba Research we consider this to be sufficient due to the low density of positive cells (less than 5% of total cells).
How do you proceed with an antigen stability test?
To assess the stability of targets in the Histoprofile®T-reg light protocol, performing a kinetic experiment necessary to analyze the same aging sample over a period of time (again up to three months). This test is typically performed using two samples for each sign, with different ranges of signal expression.
Over time, the resulting images of the NSCLC specimen were evaluated using the Halo image analysis software. Slices included in the stability test were analyzed using Halo software to determine the cell density of each multiplex target.
What are the main biomarkers that indicate effective treatment?
T-cell infiltration into a tumor is a key biomarker for estimating response to checkpoint immunotherapies. In one particular study, five colorectal cancer (CRC) samples were stained using Histoprofile®T-reg light panel and then analyzed with Halo to determine the density of CD8 T cells and Treg cells in the tumor and stroma compartments, and the density of cell clusters at the tumor/stroma border.
In this particular case, there were significantly more Tregs in the stroma than in the tumor (p = 0.026). Similarly, there were more T cells in the stromal compartment than in the tumor but not to a significant level (p = 0.096). An accumulation of T cells and Tregs is observed on the stromal side of the interface.
What makes Histoprofile®The T-reg multiplexing protocol is an outstanding tool for studying T-cell populations.
After demonstrating specificity and apparent sensitivity, the analytical performance of Histoprofile®A T-reg light protocol using anti-CD8, anti-CD3, and anti-FoxP3 indicated the ability to detect Tcyto and Treg cells in a range of solid tumors including, but not limited to, lung, head and neck, breast, and colorectal carcinomas. FFPE wipes. The protocol met acceptance criteria with respect to reproducibility and assay reproducibility.
Antigen stability shows that it is possible to detect antigens at a level similar to fresh sections after three months. In Serpa Research, Histoprofile®The T-reg light protocol has been validated on solid tumors at the experimental endpoint level.
The spatial analysis of the protocol provides an in-depth analysis of the tumor microenvironment, allowing quantification of immune cell infiltration into the tumor. We believe that Histoprofile®The T-reg light panel is a practical tool that facilitates the investigation of T-cell populations in various human solid tumors in clinical trials.
Figure 1. Comparison of cell density between simple and multiple slides. Image credit: Cerba Research
Figure 2. Evaluation of the specificity of healthy FFPE tonsil samples. Representative images of two tonsil samples stained with Histoprofile®Tregs light panel. CD3 (red), CD8 (orange), and FoxP3 (green). Scale bar: 50 µm. Image credit: Cerba Research
Figure 3. A heatmap of qualitative results obtained on healthy tonsil blocks and healthy multi-organ thermomechanical analysis was obtained. Count represents the number of samples stained for each tissue and each target in each percentage of the positive cell range. Image credit: Cerba Research
Figure 4. Heatmap of sensitivity results obtained on mass and multi-organ TMA. Count represents the number of samples stained for each tissue and each target in each percentage of the positive cell range. Image credit: Cerba Research
Figure 5. Representative images from histoprofile fidelity assessment®Light-T-reg protocol on NSCLC, TNBC, HNSCC, and CRC. CD3 (red), CD8 (orange), and FoxP3 (green). Scale bar = 100 µm. Image credit: Cerba Research
Figure 6. Histogram distribution of reproducibility test slides. Densities of CD3 (top row), CD8 (middle row), and FoxP3 (bottom row) cells in the sample post and indices. Image credit: Cerba Research
Figure 7. Assessment of CD3/CD8/FoxP3 stability on NSCLC. From top to bottom: merge, CD3 (red), CD8 (orange), FoxP3 (green). Scale bar = 100 µm. Image credit: Cerba Research
Figure 8. Representative images from spatial analysis using HaLo (the green line represents the tumor/stroma interface. Left image: Histoprofile®– Tregs CD3 (red), CD8 (orange), FoxP3 (green) light panel. Middle image: Halo masks T cells (top) and Tregs (bottom). Right image: Halo representation of the regions assessed during interface analysis. Each color represents a distance of 10 µm. Scale bar = 100 µm. Image credit: Cerba Research
Figure 9. Box plot of the density of Tregs (top) and T cells (bottom) in the Stroma (blue) and Tumor (red) of five CRC samples. Image credit: Cerba Research
Figure 10. Area diagram showing infiltration of Tregs (red) and T cells (blue) in the tumor (left) of the stroma (right) of five CRC samples. Image credit: Cerba Research
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