Immunohistochemistry (IHC) has been considered the gold standard in pathology for many years, but it allows the detection of only a single marker per tissue section. Assessing multiple markers at once is therefore challenging, as tissue is often limited, especially when additional molecular tests are required.
Multiplex immunofluorescence (IF), on the other hand, enables the parallel analysis of multiple markers. It reveals which proteins are expressed exclusively in tumor cells, exclusively in immune cells, or in both, and provides information on the density and patterns of immune cell infiltration. With CancerIFP, we offer a multiplex immunofluorescence panel that specifically examines markers of key immune checkpoints and tumor-associated signaling pathways linked to tumor progression and the immune response. This marker panel provides insights into the tumor microenvironment, potentially tumor-driving signaling pathways, and allows for verification of genetic findings at the protein level.
CancerIFP can be ordered in addition to our comprehensive molecular tumor profiling services, CancerPrecision® and CancerNeo®.
What We Offer with This Service
Our Promise to You
Service Details
- Multiplex immunofluorescence staining of 9 markers (CD45, PD-L1, LAG3, p-mTOR, p-ERK, p16, p53, HER2, TROP2) on the same tissue section – learn more about the analyzed markers
- Report with quantitative indication of marker expression
- Qualitative information about the markers in the appendix
Each marker has been validated in-house and the analysis is performed using our whole slide immunofluorescence platform.
Tissue samples are examined by a pathologist or neuropathologist to assess quality and suitability of the tissue for IF stainings. The stainings are scanned on the VS200 Slide Scanner (EVIDENT) and preprocessed as well as evaluated using the OlyVIA software (EVIDENT). Quality of stainings in controls and tumor tissue is examined by our team of biologists, pathologists, and neuropathologists. Marker expression is analyzed, reported, and approved by a pathologist or neuropathologist. The IF report includes quality control information referring to the sample that was tested as well as to the stainings that were performed.
The scoring systems used for immunofluorescence (IF) markers were adapted from established standards in chromogenic immunohistochemistry (IHC), ensuring a consistent and comparable framework. IF, however, offers enhanced sensitivity and a broader dynamic range compared to IHC. While fluorescence intensities may not exhibit a strictly linear correlation with chromogenic IHC signals, extensive validation steps were undertaken — including comparison and adjustment to chromogenic IHC during antibody establishment and validation. Nonetheless, due to intrinsic methodological differences, some variation between IF and IHC results may occur.
This service is available as add-on for FFPE-based CancerPrecision® and CancerNeo® orders.
Sample Report
Our Standard Sample Requirements
Tumor Tissue (Tumor content at least 20%)
- FFPE tumor block (min. tissue size 5 x 5 x 5 mm) (recommended sample type) or
- Unstained FFPE sections (at least 2 unstained sections of 5 μm thickness*, tissue size 5 x 5 mm) on positively charged glass slides (Superfrost® Plus or equivalent)
Please keep in mind, that this service can only be ordered along with CancerPrecision® or CancerNeo® services, so please make sure the sample requirements of these tests are met as well. In case of insufficient sample quality or tumor content, the analysis might fail.
If you have more than one option of tumor samples, please get in touch with us (tumor@cegat.com), and we would be happy to assist you in choosing the optimal specimen for your patient. Details on how to ship your samples safely can be found here.
*Our protocols and controls are validated for slides of 5 µm thickness.
This Is What Makes Our Service Special
CancerIFP combines digital pathology with spatial biology, providing a targeted selection of relevant markers to visualize immune cell infiltration, checkpoint marker expression, and signaling pathway activation. By analyzing multiple markers on the same slide, we offer a tissue-conserving approach. This is particularly important when sample quantities are limited or molecular analysis is requested in parallel. All markers have been validated in-house and are stained and evaluated by our experienced team of technicians, research scientists, pathologists, and neuropathologists. Comprehensive quality control ensures reliable, reproducible results and the best possible comparability with chromogenic IHC standards. A detailed report summarizing all results and quality parameters is provided for each analyzed sample.
IHC–IF Comparison and Multiplexing
While both immunohistochemistry (IHC) and immunofluorescence (IF) are antibody-based analyses used to detect proteins in tissue sections, they differ in marker visualization and multiplexing capacity. Whereas IHC captures chromogenic signals, IF uses a fluorescence-based detection system. In routine clinical pathology, IHC typically stains one marker per tissue section. IF, in contrast, allows the simultaneous analysis of multiple targets per tissue section.
The following illustrations demonstrate the comparability of IHC and IF signals for single markers (Fig. 1 & 2) and show how multiplex immunofluorescence analysis (Fig. 3) expands diagnostic and research capabilities beyond the limits of conventional IHC.
Fig 1: HER2 on colon carcinoma sample (IHC)
Fig 2: HER2 on colon carcinoma sample (IF)
Fig 3: CD45 (green), HER2 (yellow), LAG3 (pink) on lymph node metastasis of a urothelial carcinoma sample (IF)
Fig 1: phospho-m-TOR on glioblastoma sample (IHC)
Fig 2: phospho-m-TOR on glioblastoma sample (IF)
Fig 3: CD45 (orange), p-ERK (pink), p-mTOR (green) on high-grade glioma sample (IF)
Fig 1: PD-L1 on epitheloid glioblastoma sample (IHC)
Fig 2: PD-L1 on epitheloid glioblastoma sample (IF)
Fig 3: CD45 (brown), p-mTOR (green), PD-L1 (yellow) on giant cell glioblastoma sample (IF)
Analyzed Markers – CancerIFP
List of Markers That Are Analyzed Within This Panel via Multiplex Immunofluorescence Staining (IFP-05)
CD45, PD-L1, LAG3, p-mTOR, p-ERK, p16, p53, HER2, TROP2
Further Information on the Markers
Our service includes CD45 staining to clearly distinguish immune cells from tumor cells — a crucial step for accurate interpretation of immune checkpoint markers.
- CD45 is positive in immune cells. Staining with CD45 helps to identify lymphocytes, granulocytes and macrophages. Particularly for PD-L1 assessment (see also section below) it is of relevance to distinguish positive staining in tumor cells from positive staining in immune cells.
This panel includes 2 markers, PD-L1 and LAG3, which are relevant immune checkpoint proteins.
- PD-L1 is an immune checkpoint ligand binding PD-1 receptors on T cells, thus inactivating them and inhibiting their immune reactivity. This protein can be expressed on immune as well as on tumor cells in various entities.
- LAG3 on the other hand, is an immune checkpoint receptor and mainly expressed in exhausted T cells. Exhausted T cells have decreased proliferative capacity and may lose their cytotoxicity. High expression of both markers may indicate immune evasion.
Relevance of immune checkpoints in the tumor microenvironment:
Stimulatory and inhibitory receptors regulate T-cell activation within the tumor microenvironment. The interaction of these receptors with their ligands is crucial for immune regulation and tumor immune escape. CancerIFP specifically assesses the immune checkpoint ligand PD-L1 and the immune checkpoint receptor LAG3.
Figure 4: Illustration of immune checkpoint receptors and ligands
6 markers, namely p-mTOR, p-ERK, p16, p53, HER2, TROP2, are included in this panel to address tumor driving signalling pathways.
- p-mTOR staining may provide information on PI3K/AKT/mTOR pathway activation while p-ERK staining may give insights into MAPK pathway activation. These two pathways are often constitutively active in tumor cells. These markers can be helpful in molecular tumor boards to correlate genetic alterations of PI3K/AKT/mTOR or MAPK pathway components with phosphorylation of the respective downstream components mTOR and ERK at the protein level.
- p16 is a tumor suppressor protein that is often lost in cancers. P16 loss may lead to de-inhibition of cyclin-dependent kinases and therefore, activation of cell cycle progression. P16 staining can be helpful to verify biallelic inactivation of the gene CDKN2A, which encodes p16.
- p53 is a tumor suppressor protein encoded by the TP53 gene and activated in response to DNA damage. Once activated, it induces cell cycle arrest to allow for DNA repair mechanisms, directs cell senescence and initiates apoptosis. P53 staining can be helpful to verify a TP53 alteration at the protein level and to identify tumor cells in TP53-altered entities.
- Both HER2 and TROP2 are cell surface proteins mainly known from breast cancer. However, both may be expressed in epithelial tumors of various entities. Overexpression of these proteins is occasionally seen on RNA level. In these instances, HER2 or TROP2 staining may provide insights on membrane-bound expression at the protein level.
Within our portfolio, we cover for somatic genetic variants via our CancerPrecision® analysis. Checking for overexpression of CD274 (PD-L1), ERBB2 (HER2), TACSTD2 (TROP2), components of the PI3K/AKT/mTOR and MAPK pathway or for reduced expression of CDKN2A (p16) would be possible via gene expression add on. Finally, these findings can be verified at the protein level by multiplex IF.
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