Tumor-agnostic transcriptome-based classifier identifies spatial infiltration patterns of CD8+T cells in the tumor microenvironment and predicts clinical outcome in early-phase and late-phase clinical trials.

Background: The immune status of a patient's tumor microenvironment (TME) may guide therapeutic interventions with cancer immunotherapy and help identify potential resistance mechanisms. Currently, patients' immune status is mostly classified based on CD8+tumor-infiltrating lymphocytes. An unmet need exists for comparable and reliable precision immunophenotyping tools that would facilitate clinical treatment-relevant decision-making and the understanding of how to overcome resistance mechanisms.

Translation and evaluation of a pre-clinical 5-protein response prediction signature in a breast cancer phase Ib clinical trial.

Human protein biomarker discovery relies heavily on pre-clinical models, in particular established cell lines and patient-derived xenografts, but confirmation studies in primary tissue are essential to demonstrate clinical relevance. We describe in this study the process that was followed to clinically translate a 5-protein response signature predictive for the activity of an anti-HER3 monoclonal antibody (lumretuzumab) originally measured in fresh frozen xenograft tissue. We detail the development, qualification, and validation of the multiplexed targeted mass spectrometry assay used to assess the signature performance in formalin-fixed, paraffin-embedded human clinical samples collected in a phase Ib trial designed to evaluate lumretuzumab in patients with metastatic breast cancer.

Detection of truncated HER2 forms in formalin-fixed, paraffin-embedded breast cancer tissue captures heterogeneity and is not affected by HER2-targeted therapy.

Truncated forms of HER2, previously identified in subsets of HER2-positive breast cancer, originate from proteolytic extracellular domain (ECD) cleavage or alternative translation initiation. They lack ECD but may retain intracellular domain functionality, potentially associated with unfavorable prognosis, metastasis, and decreased sensitivity to antibody-based HER2-targeted therapy. To study the distribution of truncated HER2 in breast cancer, we detected loss of membrane-bound ECD independently of its molecular origin in paraffin sections, combining multispectral unmixing of chromogenic duplex IHC for HER2 ECD and intracellular domain with advanced image analysis.

Combat or surveillance? Evaluation of the heterogeneous inflammatory breast cancer microenvironment.

Evaluation of specific lymphocyte subsets is important in understanding the microenvironment in cancer and holds promise as a prognostic parameter in invasive breast cancer. To address this, we used digital image analysis to integrate cell abundance, distance metrics, neighbourhood relationships and sample heterogeneity into comprehensive assessment of immune infiltrates. Lymphocyte and macrophage subpopulations were detected by chromogenic duplex immunohistochemistry for CD3/perforin and CD68/CD163 in samples of invasive breast cancer.

Effects of cold ischemia and inflammatory tumor microenvironment on detection of PI3K/AKT and MAPK pathway activation patterns in clinical cancer samples.

The accuracy of common markers for PI3K/AKT and MAPK pathway activation in preclinical and clinical cancer biomarker studies depends on phosphoepitope stability and changes of phosphorylation under ischemia. Herein, we define conditions under which phosphoepitope-specific duplex immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded tumor tissues reflects pathway activation in situ as accurately as possible, and identify activation patterns linked to mutational status, pathway dependency and tumor microenvironment in clinical tumor samples, cell culture and xenograft tissues. Systematically assessing robustness of pAKT, pERK1/2, pMEK1/2 and pmTOR detection and related markers in xenograft tissues exposed to ischemia, we show that control of preprocessing and ischemia times allows accurate interpretation of staining results.