spatial-profiling-protein-rna-expression-tissue-an-approach-to-fine

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

Genentech

Preclinical data suggest that signalling through the HGF-MET pathway may confer resistance to BRAF inhibition in BRAF(V600E/K) melanoma. Therefore, blockade of HGF-MET signalling might be a valid therapeutic strategy, in combination with BRAF inhibition, in BRAF(V600E/K) melanoma. The aim of this study was to investigate the clinical relevance of these observations by evaluating the survival impact of MET expression in patients with BRAF(V600E/K) advanced melanoma treated with vemurafenib.

Impact of MET expression on outcome in BRAF(V600E/K) advanced melanoma.

Activation of the MET signalling pathway is critical in regulating multiple cellular processes underlying tumourigenic growth and has represented an attractive target for therapeutic intervention in cancer. Early stage clinical studies of multiple agents targeting this pathway have been undertaken, frequently in unselected patient cohorts with variable results. Promising data in patient subgroups in these studies indicate the need for predictive biomarkers to identify the patients most likely to benefit from these therapies. In this review, we discuss the current knowledge of mechanisms of MET activation, the status of the clinical evaluation of MET-targeted therapies, the associated efforts to identify and validate biomarkers, and the considerations and challenges for potential development of companion diagnostics.

Developing biomarkers to predict benefit from HGF/MET pathway inhibitors.

The development of human cancer is a multistep process characterized by the accumulation of genetic and epigenetic alterations that drive or reflect tumour progression. These changes distinguish cancer cells from their normal counterparts, allowing tumours to be recognized as foreign by the immune system. However, tumours are rarely rejected spontaneously, reflecting their ability to maintain an immunosuppressive microenvironment. Programmed death-ligand 1 (PD-L1; also called B7-H1 or CD274), which is expressed on many cancer and immune cells, plays an important part in blocking the 'cancer immunity cycle' by binding programmed death-1 (PD-1) and B7.1 (CD80), both of which are negative regulators of T-lymphocyte activation. Binding of PD-L1 to its receptors suppresses T-cell migration, proliferation and secretion of cytotoxic mediators, and restricts tumour cell killing. The PD-L1-PD-1 axis protects the host from overactive T-effector cells not only in cancer but also during microbial infections. Blocking PD-L1 should therefore enhance anticancer immunity, but little is known about predictive factors of efficacy. This study was designed to evaluate the safety, activity and biomarkers of PD-L1 inhibition using the engineered humanized antibody MPDL3280A. Here we show that across multiple cancer types, responses (as evaluated by Response Evaluation Criteria in Solid Tumours, version 1.1) were observed in patients with tumours expressing high levels of PD-L1, especially when PD-L1 was expressed by tumour-infiltrating immune cells. Furthermore, responses were associated with T-helper type 1 (TH1) gene expression, CTLA4 expression and the absence of fractalkine (CX3CL1) in baseline tumour specimens. Together, these data suggest that MPDL3280A is most effective in patients in which pre-existing immunity is suppressed by PD-L1, and is re-invigorated on antibody treatment. 

Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients.

Part of developing therapeutics is the need to identify patients who will respond to treatment. For HER2-targeted therapies, such as trastuzumab, the expression level of HER2 is used to identify patients likely to receive benefit from therapy. Currently, chromogenic immunohistochemistry on patient tumor tissue is one of the methodologies used to assess the expression level of HER2 to determine eligibility for trastuzumab. However, chromogenic staining is fraught with serious drawbacks that influence scoring, which is additionally flawed due to the subjective nature of human/pathologist bias. Thus, to advance drug development and precision medicine, there is a need to develop technologies that are more objective and quantitative through the collection and integration of larger data sets. In proof of concept experiments, we show multiplexed ion beam imaging (MIBI), a novel imaging technology, can quantitate HER2 expression on breast carcinoma tissue with known HER2 status and those values correlate with pathologist-determined IHC scores. The same type of quantitative analysis using the mean pixel value of five individual cells and total pixel count of the entire image was extended to a blinded study of breast carcinoma samples of unknown HER2 scores. Here, a strong correlation between quantitation of HER2 by MIBI analysis and pathologist-derived HER2 IHC score was identified. In addition, a comparison between MIBI analysis and immunofluorescence-based automated quantitative analysis (AQUA) technology, an industry-accepted quantitation system, showed strong correlation in the same blind study. Further comparison of the two systems determined MIBI was comparable to AQUA analysis when evaluated against pathologist-determined scores. Using HER2 as a model, these data show MIBI analysis can quantitate protein expression with greater sensitivity and objectivity compared to standard pathologist interpretation, demonstrating its potential as a technology capable of advancing cancer and patient diagnostics.

Multiplexed ion beam imaging analysis for quantitation of protein expression in cancer tissue sections.

Cancer immunotherapies, such as atezolizumab, are proving to be a valuable therapeutic strategy across indications, including non-small cell lung cancer (NSCLC) and urothelial cancer (UC). Here, we describe a diagnostic assay that measures programmed-death ligand 1 (PD-L1) expression, via immunohistochemistry, to identify patients who will derive the most benefit from treatment with atezolizumab, a humanized monoclonal anti-PD-L1 antibody. We describe the performance of the VENTANA PD-L1 (SP142) Assay in terms of specificity, sensitivity, and the ability to stain both tumor cells (TC) and tumor-infiltrating immune cells (IC), in NSCLC and UC tissues. The reader precision, repeatability and intermediate precision, interlaboratory reproducibility, and the effectiveness of pathologist training on the assessment of PD-L1 staining on both TC and IC were evaluated. We detail the analytical validation of the VENTANA PD-L1 (SP142) Assay for PD-L1 expression in NSCLC and UC tissues and show that the assay reliably evaluated staining on both TC and IC across multiple expression levels/clinical cut-offs. The reader precision showed high overall agreement when compared with consensus scores. In addition, pathologists met the predefined training criteria (≥85.0% overall percent agreement) for the assessment of PD-L1 expression in NSCLC and UC tissues with an average overall percent agreement ≥95.0%. The assay evaluates PD-L1 staining on both cell types and is robust and precise. In addition, it can help to identify those patients who may benefit the most from treatment with atezolizumab, although treatment benefit has been demonstrated in an all-comer NSCLC and UC patient population.

Development of a PD-L1 Complementary Diagnostic Immunohistochemistry Assay (SP142) for Atezolizumab.

Optimization and standardization of immunohistochemistry (IHC) protocols within and between laboratories requires reproducible positive and negative control samples. In many situations, suitable tissue or cell line controls are not available. We demonstrate here a method to incorporate target antigens into synthetic protein gels that can serve as IHC controls. The method can use peptides, protein domains, or whole proteins as antigens, and is compatible with a variety of fixation protocols. The resulting gels can be used to create tissue microarrays (TMAs) with a range of antigen concentrations that can be used to objectively quantify and calibrate chromogenic, fluorescent, or mass spectrometry-based IHC protocols. The method offers an opportunity to objectively quantify IHC staining results, and to optimize and standardize IHC protocols within and between laboratories. (J Histochem Cytochem 58:XXX-XXX, 2019).

Synthetic Antigen Gels as Practical Controls for Standardized and Quantitative Immunohistochemistry.

Over the past decade, invention and adoption of novel multiplexing technologies for tissues have made increasing impacts in basic and translational research and, to a lesser degree, clinical medicine. Platforms capable of highly multiplexed immunohistochemistry or in situ RNA measurements promise evaluation of protein or RNA targets at levels of plex and sensitivity logs above traditional methods - all with preservation of spatial context. These methods promise objective biomarker quantification, markedly increased sensitivity, and single-cell resolution. Increasingly, development of novel technologies is enabling multi-omic interrogations with spatial correlation of RNA and protein expression profiles in the same sample. Such sophisticated methods will provide unprecedented insights into tissue biology, biomarker science, and, ultimately, patient health. However, this sophistication comes at significant cost, requiring extensive time, practical knowledge, and resources to implement. This review will describe the technical features, advantages, and limitations of currently available multiplexed immunohistochemistry and spatial transcriptomic platforms. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Article	Year
Impact of MET expression on outcome in BRAF(V600E/K) advanced melanoma. Histopathology\| PubMed ID: 23802768	2013
Developing biomarkers to predict benefit from HGF/MET pathway inhibitors. The Journal of pathology\| PubMed ID: 24105670	2014
Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature\| PubMed ID: 25428504	2014
Multiplexed ion beam imaging analysis for quantitation of protein expression in cancer tissue sections. Laboratory investigation; a journal of technical methods and pathology\| PubMed ID: 28553935	2017
Development of a PD-L1 Complementary Diagnostic Immunohistochemistry Assay (SP142) for Atezolizumab. Applied immunohistochemistry & molecular morphology : AIMM\| PubMed ID: 29346180	2019
Synthetic Antigen Gels as Practical Controls for Standardized and Quantitative Immunohistochemistry. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society\| PubMed ID: 30879407	2019
Clinical and research applications of multiplexed immunohistochemistry and in situ hybridization. The Journal of pathology\| PubMed ID: 33723864	2021

Sandra Rost

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