Conventional immunohistochemistry (IHC) is a widely used diagnostic technique in tissue pathology. However, this technique is associated with a number of limitations, including high inter‐observer variability and the capacity to label only one marker per tissue section. This review details various highly multiplexed techniques that have emerged to circumvent these constraints, allowing simultaneous detection of multiple markers on a single tissue section and the comprehensive study of cell composition, cellular functional and cell‐cell interactions. Among these techniques, multiplex Immunohistochemistry/Immunofluorescence (mIHC/IF) has emerged to be particularly promising. mIHC/IF provides high‐throughput multiplex staining and standardized quantitative analysis for highly reproducible, efficient and cost‐effective tissue studies. This technique has immediate potential for translational research and clinical practice, particularly in the era of cancer immunotherapy.

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Introduction: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-associated mortality globally. Immune-checkpoint blockade (ICB) is one of the systemic therapy options for HCC. However, response rates remain low, necessitating robust predictive biomarkers. In the present study, we examined the expression of CD38, a molecule involved in the immunosuppressive adenosinergic pathway, on immune cells present in the tumor microenvironment. We then investigated the association between CD38 and ICB treatment outcomes in advanced HCC. Methods: Clinically annotated samples from 49 patients with advanced HCC treated with ICB were analyzed for CD38 expression using immunohistochemistry (IHC), multiplex immunohistochemistry/immunofluorescence (mIHC/IF) and multiplex cytokine analysis. Results: IHC and mIHC/IF analyses revealed that higher intratumoral CD38⁺ cell proportion was strongly associated with improved response to ICB. The overall response rates to ICB was significantly higher among patients with high proportion of total CD38⁺cells compared with patients with low proportion (43.5% vs 3.9%, p=0.019). Higher responses seen among patients with a high intratumoral CD38⁺cell proportion translated to a longer median progression-free survival (mPFS, 8.21 months vs 1.64 months, p=0.0065) and median overall survival (mOS, 19.06 months vs 9.59 months, p=0.0295). Patients with high CD38⁺CD68⁺macrophage density had a better mOS of 34.43 months compared with 9.66 months in patients with low CD38⁺CD68⁺ macrophage density. CD38^hi macrophages produce more interferon γ (IFN-γ) and related cytokines, which may explain its predictive value when treated with ICB. Conclusions: A high proportion of CD38⁺ cells, determined by IHC, predicts response to ICB and is associated with superior mPFS and OS in advanced HCC.

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.

Background: CD38 is involved in the adenosine pathway, which represents one of the immunosuppressive mechanisms in cancer. CD38 is broadly expressed across immune cell subsets, including human macrophages differentiated in vitro from monocytes, but expression by tissue-resident macrophages remains to be demonstrated. Methods: Tissue samples were obtained from 66 patients with hepatocellular carcinoma (HCC) from Singapore and analyzed using immunohistochemistry. Tumor-infiltrating leukocytes (TILs) were further examined using DEPArray™, and the phenotype of freshly isolated TILs was determined using flow cytometry. Results: CD38 was frequently co-expressed with the macrophage-specific marker CD68. CD38⁺CD68⁺ macrophage density was associated with improved prognosis after surgery, while total CD68⁺ macrophage density was associated with poor prognosis. DEPArray™ analysis revealed the presence of large (>10 μm), irregularly shaped CD45⁺CD14⁺ cells that resembled macrophages, with concurrent CD38⁺ expression. Flow cytometry also revealed that majority of CD14⁺HLA-DR⁺ cells expressed CD38. Conclusion: CD38 expression was clearly demonstrated on human macrophages in an in vivo setting. The positive association identified between CD38⁺ macrophage density and prognosis may have implications for routine diagnostic work.

Regulatory T cells (Tregs) are often enriched in tumors, where their immunosuppressive function has a key role in tumor persistence and progression. In colorectal cancer (CRC), however, Tregs are frequently associated with an improved clinical outcome. Tumor-infiltrating Tregs have been shown to exhibit a distinct signature comprising the co-stimulatory molecules (OX40, 4-1BB), cytokine receptors (IL1R2, IL21R, CCR8, CD30), and co-inhibitory molecules (PD-L1, TIGIT). Here, we showed by flow cytometry that circulating CD45RO⁺Tregs from patients with CRC (n = 25) have elevated CD30 and OX40 expression compared to healthy subjects (n = 14). We identified co-expression of CD30 and OX40 on circulating CD45RO⁺Tregs using single-cell images captured by the DEPArray^™system. The frequency of CD30⁺OX40⁺CD45RO⁺Tregs was significantly higher in CRC patients than in healthy subjects (P < 0.001). Importantly, receiver operating characteristic analysis confirmed that this CD30⁺OX40⁺Treg subset could strongly discriminate between CRC patients and healthy subjects with the highest accuracy of 92.3%, an AUC of 0.92, a sensitivity of 88%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 82.35%, and a trade-off value of 3.44%, compared to other Treg subsets. Consistently, multiplex-IHC/IF of tumor-infiltrating Tregs revealed a significant association between high densities of CD30⁺OX40⁺Tregs and improved overall survival; no such association was found for other subsets. These data suggest a potential role for CD30⁺OX40⁺Tregs as a diagnostic or prognostic biomarker in CRC.